Anti-MDA5 positive dermatomyositis (MDA5+ DM) is a type of idiopathic inflammatory myopathy with positive MDA5 autoantibody, involvement of multiple organs, heterogeneous clinical spectrum of manifestations, and high mortality (1, 2). The high mortality rate means that MDA5+ DM is a substantial challenge for clinical rheumatologists. Multiple prognostic factors have been found for this myopathy (3).

Rapidly progressive interstitial lung disease (RPILD) is the major prognostic factor that can divide patients into those with and without RPILD, leading to different short-term outcomes and therapeutic needs. For MDA5+RPILD, initiating a combined immunosuppressive therapeutic regimen early in the disease course should be considered the first-line therapy, which improves morbidity and mortality (4, 5). Several treatment protocols included the combination of high-dose glucocorticoids and calcineurin inhibitors or a triple therapy adding intravenous cyclophosphamide (5), while plasma exchange can be added for refractory disease. And tofacitinib and rituximab might have a role in the therapeutic armamentarium of this challenging to treat (4, 6, 7). These protocols significantly improved early-stage patients, whereas the 6-month mortality rate remained at around 40% (3, 8). Disease outcomes and treatments remain unsatisfactory. Clinical and prognostic heterogeneity further exacerbate the complexity and uncertainty in the development of novel treatment options.

To address this heterogeneity, the identification of clinical phenotypes and disease endotypes is required. Three distinct clinical phenotypes have been proposed, according to the predominance of pulmonary, skin-articular, or vascular symptoms in MDA5+ DM (9). However, endotype identification is still lacking. Diseases such as asthma and osteoarthritis are more advanced than MDA5+ DM in disease endotyping (10–12). Unlike phenotypes, endotypes are subtypes that are classified based on cellular, immunological, molecular, or genetic biomarkers rather than on clinically observable symptoms (9, 10). The identification of endotypes could provide deeper insights into the underlying pathological mechanisms to guide clinical management and therapeutic development. Although the aetiology and pathology remain unclear, increasing evidence suggests that multiple factors, including T cells, B cells, neutrophils and macrophages, are implicated in the pathophysiology of MDA5+DM (2). The inclusion of calcineurin inhibitors (cyclosporine A, tacrolimus) in several immunosuppressive regimens indicates that targeting T cells is an effective means to treat MDA5+ DM (3, 13). In addition, recent studies showed MDA5+ DM patients with co-existing anti-Ro52 antibodies had an increased frequency of RPILD and more aggressive phenotypes, highlighting the breach of B cell tolerance likely contributes to the pathogenesis in MDA5+ DM (13, 14). B cell depletion with rituximab has been used to treat dermatomyositis (15), which could be an alternative treatment for MDA5-DM in severe and refractory cases (2). Studies have also shown that both monocytes and lymphocytes are correlated with different prognoses of MDA5+ DM (16, 17). Lower peripheral CD3⁺T cell counts were independently associated with poorer prognosis in MDA5+DM-RPILD (18), while higher peripheral CD3⁺T cell counts were associated with longer survival. The independent risk factors for RPILD in MDA5+DM patients included lymphopenia, especially decreased levels of CD3⁺CD4⁺ T cells, elevated CD5^-CD19⁺ B cells (19). But an integrated evaluation and multi-dimensional characterization of immune cell profiles have not been achieved. The peripheral immune cells mentioned above seem to be a simple and practical approach to predicting the prognosis of MDA5+ DM in clinical practice (2). So, we exploratively performed hierarchical clustering analysis based on immune cell profiles (percentages of neutrophils, monocytes, CD3^-CD16⁺CD56⁺NK cells, CD3⁺CD4⁺ T cells, CD19⁺B cells, and CD3⁺CD8⁺ T cells) in an inpatient cohort, and analyzed the clinical and prognostic features of the identified endotypes to characterize them and to fully depict their clinical and prognostic significance. Thus, our current study may provide new clues for the disease endotype classifications, immunopathogenesis and potential therapeutic targets for MDA5+ DM.

MDA5+ DM is a distinct subtype of idiopathic inflammatory myopathies. The aetiology and pathogenesis of MDA5+ DM remain elusive, with a potential contributing role for T cells, B cells, neutrophils, and natural killer cells (2). Drugs targeting T cells (calcineurin inhibitors) and B cells (anti-CD20) have shown a certain role in clinical application (2, 13), so the classification based on immune cell subsets might provide a new perspective for the individualized treatment. In our current work, we have identified three disease endotypes based on peripheral immune cell profiles in an inpatient cohort. Endotypes based on clinical accessible data give us important references. Different clinical manifestations, laboratory results, and disease outcomes have also been revealed among the endotypes. The heterogeneity of the endotypes is multi-dimensional, with the potential to change clinicians’ perspectives on MDA5+ DM and inspire new research into the pathogenesis and therapy options for patients with different endotypes.

Among the identified endotypes, Endotype1 exhibited the best prognosis with the lowest incidence of death in 3 months, inpatient death, and inpatient ILD progression; it had the highest percentage of CD4⁺ T cells and monocytes, and the lowest percentage of neutrophils. A higher incidence of periungual capillary changes, a lower incidence of RPILD, and lower leukocyte counts were also features of this endotype. RPILD is known as an important predictor of mortality (22). Leukocyte count has been reported as an independent predictor of survival in CTD-ILD (23). Periungual erythema has also been found to be a protective predictor of survival in MDA5+ DM-ILD (24). We also observed in Endotype1 with the lowest mortality at 3 months had higher periungual erythema.

Endotype2 has the highest B cells percentage among the three endotype, showing notable treatment implications. Most patients had RPILD, spontaneous pneumomediastinum, muscle involvement, and inpatient ILD progression, despite the most intensive therapy. Levels of γ-GT, ALP, and urea-to-creatinine ratio were all higher in this endotype. As γ-GT was reported to be higher in dead patients with MDA5+ DM, and as liver dysfunction might be a clinical manifestation caused by activated macrophages in MDA5+ DM (25, 26), liver function monitoring should be recommended. Further, an increase in the urea to creatinine ratio (UCR) has been reported to reflect the state of catabolism and poor nutrition in persistent critical illness and other chronic diseases (27, 28). The increase of UCR observed in this endotype might be largely due to disease severity and poor overall condition of this endotype. As the endotype mainly featured an increase in the percentage of B cells, CD3^-CD20⁺ cells, and plasma cells, B-cell -targeted therapy needs greater attention. Unfortunately, no such therapy was used in Endotype2 (shown in Table 2 ). A systematic review that presented information on 35 patients with MDA5+ DM who had received rituximab treatment found that 71.43% (25/35) of patients responded to treatment (29). The anti-CD20 drug seems to be promising, and other B-cell-targeted drugs including Bruton’s tyrosine kinase inhibitors are also worth investigating. Recently, a refractory case with a life-threatening RPILD in MDA5+ DM has been successfully treated with daratumumab, an anti-CD38-antibody (30). Single-cell RNA sequence on peripheral B cells also revealed that the B cell compartment was greatly activated with the terminal differentiation of antibody-secreting cell in active MDA5+ DM patients (13). More studies should be encouraged to explore the treatment potential of B-cell and CD38⁺ plasma-targeted therapies in MDA5+ DM, especially in the high B cell% endotype.

Endotype3 was characterized by the significantly elevated CD8⁺ T cells, having the highest level of CRP among the three groups, showing a poor prognosis that presented as higher RPILD, 3-month mortality than that in Endotype1. The expression levels of IL-2 and IL-2R, which are related to proliferative and activation of T cells (31, 32), were significantly elevated in Endotype3. Recent study also found that CD8⁺ T cell response is activated in MDA5+ DM, suggesting that CD8⁺ T cells could be the potential target (13). More cytomegalovirus and Pneumocystis infections were found in this endotype. Concomitant infection is a common complication of MDA5+ DM, which is also a potential trigger of MDA5-DM (2). Infections, such as viruses or fungi, have complex influence to immune system (2, 33). In vivo antigen-activated human T cells have been shown to be identified by a CD38 bright, HLA-DR⁺ phenotype after infection (34). Patients in Endotype3 showed the increased expression of IFN-gamma and the highest percentage of CD8⁺CD38⁺ cells and CD8⁺HLA-DR⁺ cells, which might be the complicated results of interactions between infection and primary disease. More researches are needed to explain the underlying mechanism and cause-and-effect relationship. In this state of MDA5+ DM, the cut-off of CD8⁺CD38⁺ cells and CD8⁺HLA-DR⁺ cells could distinguish ADM+ DM with or without infections, which might have a certain significance for clinical judgments and need more studies to validate. Uncontrolled expansion of CD8⁺ T cells producing IFN-gamma and other cytokines delay the termination step of the pro-inflammatory response (31). Studies have pointed out that CD38 is a surface marker of T cell exhaustion, which can inhibit the expression of cytotoxicity-related molecules through histone methyltransferase EZH2, thereby reducing the abilities of CD8⁺ T cell degranulation, perforin production and killing (35, 36), which increases the risk of infection in the body. These may further imply that both the disorder in the immune system and exposure to viral infection could trigger the deterioration of MDA5+ DM in different endotypes. Timely screening for concurrent infections and anti-infective therapies are crucial for better disease outcomes. The role of CD8⁺ T cells in MDA5+ DM requires further exploration.

This study has some limitations. Patients with missing data and those with <2500 lymphocytes for flow cytometry were not included. The need for prompt treatment and lack of untreated inpatients meant that the enrolled patients were already treated before sampling. Asymptomatic patients with no hospitalization history in our center could also be missed by the current study. It must be pointed out that our current work is mainly described in single-center, thus limiting the external validity of these findings. Further prospective studies are required to perform in other populations or diseases. However, this is a novel study that successfully identified distinct disease endotypes from immune cell profiles and fully explored their significance for the management of patients with MDA5 + DM.

In conclusion, three disease endotypes were identified through hierarchical clustering analysis of the immune cell profiles of patients with MDA5+ DM. Clinical and prognostic differences were also confirmed among the endotypes, indicating the underlying heterogeneity. These results could help promote clinical stratification, pathogenesis investigations, and personalized treatment strategy or novel drug discovery targeting such key immune cell types in MDA5+ DM.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving humans were approved by Renji Hospital, Shanghai Jiao Tong University School of Medicine. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

RG: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. YY: Data curation, Writing – original draft. LG: Resources, Writing – review & editing. XiL: Resources, Software, Writing – review & editing. YM: Data curation, Resources, Software, Writing – review & editing. XuL: Data curation, Funding acquisition, Investigation, Resources, Supervision, Writing – review & editing, Writing – original draft. LL: Funding acquisition, Investigation, Resources, Supervision, Writing – review & editing.