Association and exploratory clinical significance of peripheral blood mononuclear cell microRNA-155 with T-Cell subset imbalance in newly diagnosed paediatric immune thrombocytopenia

Abstract

Objective

To investigate the association of peripheral blood mononuclear cell micro RNA-155 miR-155 expression with T-cell subset imbalance and disease severity in newly diagnosed paediatric immune thrombocytopenia (ITP).

Methods

Eighty-seven children with newly diagnosed ITP (43 severe, 44 non-severe) and 40 healthy controls were enrolled. Peripheral blood mononuclear cell miR-155 expression was measured via quantitative reverse transcription polymerase chain reaction; Th1, Th2, Th17 and regulatory T (Treg) cell proportions were detected using whole-blood flow cytometry and expressed as percentages of gated CD3⁺ CD4⁺ T cells. Associations and exploratory predictive performance were analysed using Pearson correlation and receiver operating characteristic curves.

Results

Peripheral blood mononuclear cell miR-155 expression was significantly higher in patients with ITP than in controls and higher in severe than in non-severe ITP (P < 0.05); Th1 and Th17 cells, as well as Th1/Th2 and Th17/Treg ratios, were increased, whereas Th2 and Treg cells were decreased in ITP (P < 0.05); miR-155 expression was positively associated with Th1, Th17, Th1/Th2 and Th17/Treg and negatively associated with Th2, Treg and platelet count (P < 0.05). The exploratory combined model of miR-155, Th1/Th2 and Th17/Treg yielded an area under the curve of 0.928 (95%CI: 0.865–0.973) for severe ITP, with 79.10% sensitivity and 95.50% specificity.

Conclusion

Peripheral blood mononuclear cell miR-155 expression is associated with T-cell subset imbalance and disease severity in newly diagnosed paediatric ITP. The combined immune–cellular indicator may support severe-risk stratification, but prospective validation and functional studies are required.

Introduction

Immune thrombocytopenia (ITP) is a common acquired autoimmune haemorrhagic disorder in children, characterised by immune-mediated platelet (PLT) destruction and impaired PLT production, with an estimated incidence of approximately 2–5 per 100,000 persons [1]. Although many paediatric cases undergo spontaneous remission, describing paediatric ITP as simply benign is clinically incomplete because even transient disease may be accompanied by mucocutaneous bleeding, activity restriction, fatigue, treatment-related adverse effects, anxiety and impaired quality of life [1, 2]. Approximately 20%–30% of children may develop persistent or chronic ITP, and a subset has a poor response to first-line therapy or clinically meaningful bleeding risk [3]. According to the ‘Diagnosis and Treatment Recommendations for Primary Immune Thrombocytopenia in Children (2019 Edition)’ [4], newly diagnosed ITP refers to a disease duration of 3 months or less. In the present study, severe ITP was defined as PLT count < 20 × 10⁹/L accompanied by clinically significant mucocutaneous haemorrhage or requiring urgent clinical attention, whereas non-severe ITP was defined as newly diagnosed ITP with PLT count ≥ 20 × 10⁹/L and absent or mild self-limiting mucosal bleeding. Because treatment intensity and early monitoring differ between these clinical phenotypes, identifying immune indicators associated with severe presentation remains relevant to risk stratification and individualised management.

The pathogenesis of ITP has not been fully elucidated, but immune dysregulation is generally regarded as a core mechanism [5, 6]. This dysfunction involves immune-mediated PLT destruction, impaired megakaryocyte maturation and PLT production and abnormal cellular immunity mediated by T and B lymphocytes; T lymphocytes are central components of adaptive immunity and include CD4⁺ helper T cells and CD8⁺ cytotoxic T cells, both of which can participate in PLT-directed autoimmunity [7]. Furthermore, CD4⁺ T cells can differentiate into effector subsets such as Th1, Th2 and Th17 cells, whereas regulatory T cells (Treg) maintain immune tolerance by suppressing excessive immune activation. Under physiological conditions, Th1/Th2 and Th17/Treg axes remain dynamically balanced. In children with ITP, available evidence suggests a shift towards enhanced Th1 and Th17-associated inflammation and relatively insufficient Treg-mediated tolerance; however, the degree to which these changes reflect disease severity requires additional stratified clinical analysis [8].

MicroRNAs (miRNAs) are short non-coding RNA molecules that regulate cell proliferation, differentiation, apoptosis and immune responses through post-transcriptional regulation of target mRNAs. MiR-155 is one of the most extensively studied immune-related miRNAs and is upregulated in activated immune cells, including monocytes/macrophages and lymphocyte subsets [9]. Increased miR-155 expression has been reported in autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis and has been associated with inflammatory activity [10, 11]. In ITP, previous studies have reported increased miR-155 expression and have suggested associations with immune imbalance, SOCS1/SHIP1-related signalling, macrophage polarisation and PLT destruction [12, 13]. Nevertheless, most existing studies have not clearly separated paediatric ITP from adult ITP or focused on the newly diagnosed stage with severity stratification. The present cross-sectional study therefore evaluates peripheral blood mononuclear cell (PBMC) miR-155 expression, T-cell subset proportions and their associations in newly diagnosed paediatric ITP, with an emphasis on severe versus non-severe subgroups and exploratory combined receiver operating characteristic (ROC) modelling. The study was designed to generate clinically relevant associative evidence rather than to establish direct molecular causality.

Materials and methods

Study participants

A total of 87 children with newly diagnosed ITP admitted to the Department of Pediatrics at the First Affiliated Hospital of Henan Medical University between January 2021 and June 2025 were consecutively enrolled at the time of initial admission before ITP-specific treatment, including glucocorticoids, intravenous immunoglobulin, anti-D immunoglobulin, rituximab, thrombopoietin receptor agonists, other immunosuppressive agents or PLT transfusion. Treatment exposure before sampling was verified through admission medical records and guardian medication history, and patients with any documented prior ITP-specific treatment before index sampling were excluded. The upper age limit of 14 years was selected because it corresponds to the paediatric admission scope of the study hospital and the paediatric age range used in the Chinese paediatric ITP diagnostic and treatment specification [4]. The inclusion criteria were as follows: (1) aged 0–14 years; (2) meeting the diagnostic and staging criteria for primary ITP specified in the ‘Standards for the Diagnosis and Treatment of Primary Immune Thrombocytopenia in Children (2019 Edition)’ [4], including PLT count < 100 × 10⁹/L in at least two routine blood tests, isolated thrombocytopenia with skin petechiae or ecchymoses, exclusion of diseases causing secondary thrombocytopenia and absent or only slight splenomegaly; (3) disease duration ≤ 3 months; and (4) informed consent provided by guardians. Severe ITP was defined as PLT count < 20 × 10⁹/L with clinically significant mucocutaneous haemorrhage, and non-severe ITP was defined as PLT count ≥ 20 × 10⁹/L with skin petechiae, ecchymoses or mild self-limiting mucosal haemorrhage. Platelet count was measured on the day of diagnosis and before disease-specific therapy. Secondary thrombocytopenia was excluded through standardised clinical assessment, repeated complete blood count, peripheral blood smear review, liver and renal function tests, coagulation profile, autoimmune screening when indicated, viral infection screening when clinically indicated and bone marrow examination when peripheral smear, blood cell lineage abnormalities, organomegaly or systemic symptoms suggested alternative haematologic disease. Children with acute infection, acute inflammatory disease, vaccination within the past 4 weeks, systemic autoimmune disease, malignant tumour, severe organ dysfunction, aplastic anaemia, leukaemia, myelodysplastic syndrome, hereditary thrombocytopenia or prior intravenous immunoglobulin exposure before index sampling were excluded. Forty healthy children who underwent routine physical examination during the same period were selected as the control group by frequency matching for age and sex. Controls were not hospitalised and had no history or clinical evidence of acute infection, chronic inflammatory disease, autoimmune disease, malignancy, thrombocytopenia or immunomodulatory therapy. There were no statistically significant differences in gender, age and body mass index (BMI) between the ITP and control groups (P > 0.05), as shown in Table 1. This study was conducted in accordance with the Declaration of Helsinki and approved by the Medical Research Ethics Committee of the First Affiliated Hospital of Henan Medical University (Approval Number: EC-LW026-051).

Table 1.

Comparison of general clinical data and routine blood parameters among the three groups (x ± s)

Parameter	Control group (n = 40)	Non-severe ITP group (n = 44)	Severe ITP group (n = 43)	P value
Sex (male/female)	21/19	26/18	25/18	0.879
Age (years; mean ± SD)	4.50 ± 3.42	4.79 ± 3.58	5.39 ± 3.50	0.501
BMI (kg/m²)	17.49 ± 1.71	16.13 ± 2.54	17.18 ± 2.75	0.058
WBC (×10⁹/L)	7.85 ± 2.65	7.93 ± 2.94	8.61 ± 3.03	0.427
RBC (×10¹²/L)	4.60 ± 0.25	4.35 ± 0.42	4.31 ± 0.43	0.001
Hb (g/L)	124.70 ± 10.36	118.22 ± 11.30	118.28 ± 12.35	0.013
PLT (×10⁹/L)	350.61 ± 85.50	42.12 ± 22.67*	6.28 ± 4.71*#	< 0.0001

Age was summarized as mean ± SD; the predefined eligibility age range was 0–14 years

^#P < 0.05 vs. non-severe group. Sex comparison was performed by χ² test; other parameters were compared by ANOVA followed by LSD-t test

*P < 0.05 vs. control group

Based on the results of the preliminary experiment and previous literature reports [12, 13], the difference in PBMC miR-155 expression between the ITP group and the control group was used as the primary index for calculation. With a two-sided α = 0.05 and a test power of 1-β = 0.90, the expected sample size ratio between the ITP group and the control group was 2:1. Considering a 10% sample disqualification rate, it was calculated that at least 86 cases in the ITP group and 40 cases in the control group were required. In this study, 87 cases in the ITP group and 40 in the control group were enrolled, meeting the sample size requirement.

Main reagents and instruments

The main reagents and instruments were incorporated into the experimental procedures. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays used a real-time fluorescence quantitative PCR kit (Takara Bio Inc., Shiga, Japan), total RNA extraction kit (Omega Bio-tek, Norcross, GA, USA), reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA), miR-155-specific primers and U6 small nuclear (sn)RNA reference primers (Sangon Biotech, Shanghai, China) and an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Flow cytometry used CD4-FITC, CD183-FITC, CD196-PE, CD3-ECD, CD4-PC7, CD3-PE, CD25-ECD, CD127-PC5.5, CD45-PC7 and OptiLyse C lysing solution from Beckman Coulter (Brea, CA, USA) on a Beckman Coulter DxFLEX flow cytometer. Complete blood counts were measured using a Sysmex XN-9000 automatic haematology analyser (Kobe, Japan), with centrifugation performed using an Eppendorf 5810R high-speed refrigerated centrifuge (Hamburg, Germany) and RNA concentration and purity measured using a Thermo Fisher Scientific ultraviolet spectrophotometer.

Experimental methods

Sample collection and processing

Fasting peripheral venous blood (6 mL) was collected from all participants in the early morning before ITP-specific treatment. Before venipuncture, treatment-naive status was confirmed by reviewing admission medication records and guardian-reported medication history. Each sample was aliquoted into three ethylenediaminetetraacetic acid-anticoagulant tubes (2 mL each): Tube 1 for complete blood cell analysis and PLT detection, Tube 2 for whole-blood flow cytometry and Tube 3 for PBMC isolation and RNA extraction for miR-155 detection. All samples were processed within 2 h of collection to ensure biological stability. Clinical screening for fever, respiratory or gastrointestinal infection, recent vaccination and other acute inflammatory conditions was completed before sample inclusion.

Routine blood index detection

Clinical data, including gender, age, BMI, bleeding presentation at diagnosis, treatment exposure before sampling and exclusion-screening information, were collected from the hospital electronic medical record system. Routine blood indices, including white blood cell count (WBC), red blood cell count (RBC), haemoglobin (Hb) and PLT count, were detected using the Sysmex XN-9000 automatic haematology analyser following standardised operating procedures. Age was recorded as a continuous variable in years and summarised as mean ± standard deviation; the eligibility age range was 0–14 years.

Quantitative reverse transcription polymerase chain reaction detection of miR-155 expression

Peripheral blood mononuclear cell isolation was performed as follows. First, 2 mL of anticoagulated blood was diluted with phosphate-buffered saline (PBS), layered onto lymphocyte separation medium, centrifuged at 1,500 rpm for 20 min at 20 °C and then at 2,000 rpm for 10 min. The buffy coat was aspirated, washed twice with PBS (1,500 rpm, 10 min), resuspended in TRIzol™ reagent, and stored at − 80 °C. Before use, samples were thawed rapidly at 37 °C. Total RNA was extracted from PBMCs using an RNA extraction kit; RNA purity (A260/A280 = 1.8–2.0) and concentration were detected via ultraviolet spectrophotometry, and integrity was verified using 1% agarose gel electrophoresis. Complementary (c)DNA was synthesised from total RNA using a reverse transcription kit under the following conditions: 37 °C for 15 min, 85 °C for 5 s and storage at 4 °C. The 20-µL qRT-PCR reaction system contained 10 µL of SYBR™ Green Mix, 0.5 µL of upstream/downstream primer, 2 µL of cDNA template and 7 µL of nuclease-free water. Amplification conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s; melting curve analysis was performed at 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. All reactions were performed in triplicate; U6 snRNA was used as the internal reference, and miR-155 relative expression was calculated using the 2^−ΔΔCt method [14]. Peripheral blood mononuclear cells were selected to evaluate cellular immune-associated miR-155 expression; because PBMCs contain monocytes, B cells, NK cells and T cells, the PBMC signal was not interpreted as T cell–specific expression. The primer sequences are shown below:

• miR-155 upstream: 5’-TTTAATGCTAATCGTGATAGGGGT-3’.

• miR-155 downstream: 5’-GGTATCCAGTGCAGGGTCCGAGGT-3’.

• U6 upstream: 5’-CTCGCTTCGGCAGCACA-3’.

• U6 downstream: 5’-AACGCTTCACGAATTTGCGT-3’.

Flow cytometry detection of T-cell subsets

Flow cytometry was performed using a standardised whole-blood erythrocyte-lysis protocol rather than PBMC-based immunophenotyping. Three flow cytometry tubes (A, B and C) were prepared with 100 µL of anticoagulated whole blood in each. Tube A (for Th1/Th2/Th17 detection) contained 10 µL each of CD3-ECD, CD4-PC7, CD183-FITC and CD196-PE antibodies; phenotypes were defined as Th1 (CD3⁺CD4⁺CD183⁺CD196⁻), Th2 (CD⁺CD4⁺CD183⁻CD196⁻) and Th17 (CD3⁺CD4⁺CD183⁻CD196⁺). Tube B (for Treg detection) contained 10 µL each of CD3-PE, CD4-FITC, CD25-ECD, CD127-PC5.5 and CD45-PC7 antibodies; Treg cells were defined as CD3⁺CD4⁺CD25⁺CD127^low/-. Tube C contained corresponding isotype controls. After gentle vortexing, samples were incubated at 20–25 °C for 20 min in the dark, mixed with 500 µL of OptiLyse C lysing solution and incubated for 15 min in the dark until complete haemolysis. After washing with 2 mL of PBS (1,500 rpm, 5 min), the pellet was resuspended in 500 µL of PBS and analysed using the Beckman Coulter DxFLEX flow cytometer within 2 h. A minimum of 10,000 gated lymphocyte events were collected per sample, and samples that failed the acquisition threshold were repeated. Lymphocytes were first gated by forward scatter and side scatter, followed by CD3⁺CD4⁺ T-cell gating; Th1, Th2, Th17 and Treg percentages were expressed as proportions of gated CD3⁺CD4⁺T cells, not as proportions of total PBMCs; Th1/Th2 and Th17/Treg ratios were calculated from these CD3⁺CD4⁺ T-cell proportions. The gating strategy is shown schematically in Fig. 1.

Fig. 1.

Schematic flow cytometric gating strategy for CD4⁺ T-cell subset analysis. Whole blood was processed by erythrocyte lysis, lymphocytes were selected by FSC/SSC, and CD3⁺CD4⁺ T cells were then gated. Th1, Th2, Th17, and Treg populations were independently identified from the gated CD3⁺CD4⁺ T-cell population. Th1, Th2, and Th17 cells were classified using CD183 and CD196, and Treg cells were classified as CD3⁺CD4⁺CD25⁺CD127^low/-. Percentages were calculated among gated CD3⁺CD4⁺ T cells

The flow cytometry protocol was designed for proportional immunophenotyping. Absolute subset counts were not generated because single-platform counting beads and simultaneously measured lymphocyte subset absolute counts were not incorporated into the pre-specified assay workflow.

Statistical analysis

Data were collated in Excel and analysed using SPSS 26.0. Measurement data were expressed as mean ± standard deviation (x ± s). An independent-samples t-test was used for two-group comparisons. For multiple-group comparisons, normality (Shapiro–Wilk test) and variance homogeneity (Levene test) were assessed first: one-way analysis of variance with a least significant difference t-test was used for normal and homogeneous data, and a Kruskal–Wallis H test with a Mann–Whitney U test and Bonferroni correction were used for non-homogeneous data. Enumeration data were expressed as rate (%) and compared using the chi-square (χ²) test. Pearson correlation analysis was used to explore linear associations between variables; these analyses were interpreted as associative and not causal. Receiver operating characteristic curve analysis was performed to evaluate diagnostic or predictive performance, with calculations of area under the curve (AUC), 95%CI, sensitivity, specificity and Youden index. For the combined indicator, a binary logistic regression model was fitted with severe versus non-severe ITP as the dependent variable and miR-155, Th1/Th2 and Th17/Treg as predictors. The predicted probability from this model was used for combined ROC analysis; AUCs were compared using the DeLong method. Because the study was cross-sectional and single-centre, ROC analyses were considered exploratory. A P-value < 0.05 was considered statistically significant.

Results

General clinical data and routine blood indices

There were no significant differences in gender, age, BMI or WBC among the control group, non-severe ITP group and severe ITP group (P > 0.05). Moreover, RBC and Hb showed statistically significant but clinically small intergroup differences, and all enrolled children met the predefined requirement of no alternative haematologic disorder or abnormal non-PLT blood cell lineage suggesting secondary thrombocytopenia. No enrolled child had clinical evidence of acute infection or acute inflammatory disease at the time of sampling. Platelet count differed significantly among the three groups (P < 0.001): PLT in both ITP subgroups was significantly lower than that in the control group (P < 0.001), and PLT in the severe ITP group was significantly lower than that in the non-severe group (P < 0.001) (Table 1).

MiR-155 expression and T-cell subset proportions

Peripheral blood mononuclear cell miR-155 relative expression in the ITP group was significantly higher than that in the control group, and expression in the severe ITP group was significantly higher than that in the non-severe group (P < 0.05). Compared with the control group, the ITP group had significantly increased Th1 and Th17 proportions and Th1/Th2 and Th17/Treg ratios, whereas Th2 and Treg proportions were significantly decreased (P < 0.05). All flow cytometry percentages in Table 2 refer to proportions of gated CD3⁺CD4⁺ T cells. The imbalance was more pronounced in the severe ITP group than in the non-severe group (P < 0.05) (Table 2).

Table 2.

Comparison of CD3⁺CD4⁺T-cell subset proportions and PBMC miR-155 expression among the three groups (x ± s)

Parameter	Control group (n = 40)	Non-severe ITP group (n = 44)	Severe ITP group (n = 43)	P value
Th1 (%)	10.96 ± 2.69	15.48 ± 3.00*	20.15 ± 3.25*#	< 0.0001
Th2 (%)	84.02 ± 4.27	74.49 ± 4.11*	68.44 ± 5.14*#	< 0.0001
Th1/Th2	0.13 ± 0.04	0.21 ± 0.05*	0.30 ± 0.07*#	< 0.0001
Th17 (%)	2.88 ± 1.02	5.95 ± 1.04*	7.74 ± 1.59*#	< 0.0001
Treg (%)	8.02 ± 1.10	7.14 ± 1.13*	6.47 ± 1.19*#	< 0.0001
Th17/Treg	0.37 ± 0.15	0.84 ± 0.18*	1.24 ± 0.35*#	< 0.0001
miR-155	0.81 ± 0.20	3.71 ± 0.91*	5.56 ± 1.08*#	< 0.0001

Th1, Th2, Th17, and Treg values are expressed as percentages of gated CD3⁺CD4⁺ T cells

*P<0.05 vs. control group; # P<0.05 vs. non-severe ITP group

Correlation analysis of miR-155 expression with T-cell subsets and platelet count

Pearson correlation analysis showed that PBMC miR-155 relative expression in newly diagnosed paediatric ITP was positively associated with Th1 proportion (r = 0.725, P < 0.001), Th17 proportion (r = 0.789, P < 0.001), Th1/Th2 ratio (r = 0.698, P < 0.001) and Th17/Treg ratio (r = 0.812, P < 0.001). Peripheral blood mononuclear cell miR-155 expression was negatively associated with Th2 proportion (r = − 0.654, P < 0.001), Treg proportion (r = − 0.763, P < 0.001) and PLT (r = − 0.835, P < 0.001). These findings indicate coordinated associations between PBMC miR-155 expression, CD3⁺CD4⁺ T-cell subset imbalance and thrombocytopenia, but do not establish direct regulatory effects (Fig. 2).

Fig. 2.

Correlation between PBMC miR-155 expression and T-cell subset proportions and platelet count in newly diagnosed pediatric ITP. T-cell subsets are expressed as percentages of gated CD3⁺CD4⁺ T cells. Panels are arranged as Th1, Th2, Th1/Th2 ratio, Th17, Treg, Th17/Treg ratio, and platelet count

Exploratory predictive value of miR-155 and T-cell subsets for severe disease in children with newly diagnosed immune thrombocytopenia

Receiver operating characteristic curve analysis showed that the logistic regression–derived combination of miR-155 + Th1/Th2 + Th17/Treg had the highest exploratory discrimination for severe ITP, with an AUC of 0.928 (95%CI: 0.865–0.973), sensitivity of 79.10% and specificity of 95.50%. The combined model showed a higher AUC than any single indicator (P < 0.05), but this result should be interpreted as hypothesis-generating because no external prospective validation cohort was included (Table 3; Fig. 3).

Table 3.

Exploratory predictive value of miR-155 and T-cell subset ratios for severe disease in children with newly diagnosed ITP

Parameter	AUC	95% CI	Youden	Cutoff	Sensitivity (%)	Specificity (%)
miR-155	0.863	0.778 ~ 0.931	0.560	4.980	62.80	93.20
Th17/Treg	0.836	0.747 ~ 0.910	0.589	0.890	90.70	68.20
Th1/Th2	0.797	0.694 ~ 0.884	0.429	0.210	88.40	64.50
Combined	0.928abc	0.865 ~ 0.973	0.745	0.592	79.10	95.50

Combined indicator was generated from a binary logistic regression model including miR-155, Th1/Th2, and Th17/Treg. AUC comparisons were performed by DeLong test

^aP<0.05 vs. miR-155

^bP<0.05 vs. Th17/Treg

^cP<0.05 vs. Th1/Th2

Fig. 3.

Exploratory ROC analysis comparing miR-155, Th17/Treg ratio, Th1/Th2 ratio, and the logistic-regression-derived combined indicator for severe disease in newly diagnosed pediatric ITP

Discussion

This cross-sectional study evaluated PBMC miR-155 expression and CD3⁺CD4⁺ T-cell subset imbalance in newly diagnosed paediatric ITP and compared severe and non-severe subgroups. The results showed that PBMC miR-155 expression was increased in ITP compared with controls and was higher in severe than non-severe ITP. Children with ITP also showed a Th1/Th17-skewed and Treg-reduced immune profile, as reflected by increased Th1 cells, Th17 cells, Th1/Th2 ratio and Th17/Treg ratio and decreased Th2 and Treg cells. These findings are consistent with previous reports that T cell–mediated immune dysregulation and miR-155–associated inflammatory signalling participate in ITP immunopathology [8, 12, 13]. The main contribution of this study is therefore not the discovery of miR-155 upregulation itself, but the severity-stratified paediatric analysis at the newly diagnosed stage and the exploratory evaluation of a combined immune–cellular indicator for severe presentation.

Characteristics of T-cell subset imbalance in newly diagnosed paediatric immune thrombocytopenia (severe/non-severe)

The balance of T-cell subsets is critical for maintaining immune tolerance, and its disruption is associated with autoimmune disease activity. In this study, newly diagnosed paediatric ITP was characterised by increased proportions of Th1 and Th17 cells and decreased proportions of Th2 and Treg cells. This imbalance was more pronounced in the severe subgroup, in which the Th17/Treg ratio was markedly higher and the Treg proportion was lower than in the non-severe subgroup. These results support the interpretation that a more prominent inflammatory CD4⁺ T-cell profile is associated with severe presentation. Mechanistically, interferon-gamma produced by Th1 cells can support cytotoxic immune activation, and interleukin (IL)-17 produced by Th17 cells can amplify inflammatory responses, whereas Treg cells suppress effector T-cell activation through mediators such as IL-10 and transforming growth factor-beta [15]. In this study, the proportion of Treg cells in the severe subgroup was 6.47%±1.19%, lower than in the non-severe subgroup 7.14%±1.13%, suggesting that insufficient Treg representation may be an immune feature of severe ITP. Recent studies and reviews have also emphasised that T-cell dysfunction and impaired immune tolerance are relevant to ITP severity and treatment response [16, 17]. However, because the present study used a cross-sectional design, these subset differences should be interpreted as severity-associated immune signatures rather than proof that one subset directly causes the severe phenotype.

Peripheral blood mononuclear cell miR-155 expression and its association with T-cell subset imbalance in newly diagnosed paediatric immune thrombocytopenia (severe/non-severe)

As an immune-regulatory miRNA, miR-155 is linked to the activation of inflammatory immune pathways [9]. The present study found that PBMC miR-155 expression was higher in newly diagnosed paediatric ITP and was further increased in severe disease. Correlation analysis showed that PBMC miR-155 expression was positively associated with Th1 and Th17 proportions and negatively associated with Th2 and Treg proportions. These results are compatible with the hypothesis that miR-155-related immune activation accompanies Th1/Th17 polarisation and reduced regulatory balance in ITP. Experimental studies in immune cells have shown that miR-155 can target negative regulatory molecules such as SOCS1 and SHIP1, thereby affecting JAK/STAT and PI3K/Akt signalling pathways [18–20]. Nevertheless, the current study measured miR-155 in bulk PBMCs rather than purified CD4⁺ T cells; PBMC miR-155, therefore, reflects a composite cellular signal from monocytes, B cells, NK cells and T cells. The observed correlations with T-cell subsets are biologically plausible and clinically informative, but they do not prove that miR-155 directly regulates T-cell differentiation in these patients. In vitro overexpression or silencing experiments in purified T cells are required to confirm T cell-intrinsic mechanisms. Because viable purified T-cell samples were not retained for cell-culture manipulation, such functional assays could not be added in the current revision.

Exploratory predictive value of miR-155 Combined with T-cell subsets for severe disease in newly diagnosed paediatric immune thrombocytopenia

Early identification of severe risk in newly diagnosed paediatric ITP is clinically important because management decisions depend not only on PLT count but also on bleeding, comorbidities, access to follow-up and patient-centred factors [1, 2]. The ROC curve analysis in this study showed that the combination of PBMC miR-155, Th1/Th2 ratio and Th17/Treg ratio yielded an AUC of 0.928 for severe ITP, with 79.10% sensitivity and 95.50% specificity. This finding suggests that integrating a cellular miRNA indicator with CD4⁺ T-cell subset ratios may improve discrimination compared with a single immune variable. However, the model was developed and tested in a single-centre cohort; therefore, the predictive performance is exploratory and should not be interpreted as a validated clinical decision rule. Before clinical deployment, the combined model requires prospective validation, calibration assessment, external testing and evaluation against routine clinical predictors such as bleeding score, PLT count trajectory and treatment response.

Study limitations and future perspectives

This study has several limitations. First, it was a single-centre, cross-sectional study with a limited sample size, which may introduce selection bias and precludes assessment of temporal changes. Second, PBMC miR-155 was measured in bulk PBMCs, so the source-cell contribution of monocytes, B cells, NK cells and T cells could not be separated; purified CD4⁺ T cell miR-155 assays would be more appropriate for mechanistic inference. Third, plasma or serum miR-155 was not measured, and the present data therefore support PBMC miR-155 as a cellular–immune indicator rather than a cell-free systemic biomarker. Fourth, in vitro functional assays of miR-155 overexpression or silencing were not performed, meaning causal regulatory effects on T-cell subsets could not be demonstrated. Because viable purified T-cell samples were not retained for cell-culture manipulation, these assays could not be added retrospectively in the current revision. Fifth, the flow cytometry protocol quantified subset proportions rather than absolute subset counts, and validated bleeding scores or longitudinal samples were not available for correlation with specific bleeding patterns, treatment response, persistence or chronicity. Patient follow-up outcome data were not systematically collected, so follow-up-based response, persistence, chronicity and longitudinal miR-155 and cell subsets changes could not be added to this revision. Future studies should include prospective multicentre cohorts, external validation of the combined ROC model, paired PBMC and plasma/serum miR-155 measurements, purified CD4⁺ T cell functional experiments, standardised bleeding scores, absolute lymphocyte subset quantification and age-stratified analyses.

Conclusion

Peripheral blood mononuclear cell miR-155 expression is upregulated in newly diagnosed paediatric ITP and is higher in severe than in non-severe disease. Peripheral blood mononuclear cell miR-155 expression is associated with Th1/Th2 and Th17/Treg subset imbalance and with PLT count, suggesting that it reflects the inflammatory immune status of newly diagnosed paediatric ITP. The combination of PBMC miR-155, Th1/Th2 ratio and Th17/Treg ratio showed high exploratory discrimination for severe ITP in this cohort. These findings support the potential value of integrated immune-cellular assessment for risk stratification, although prospective validation and functional mechanistic studies are required before the combined indicator can be applied as a clinical biomarker or therapeutic target.

Authors’ contributions

Conception and design of the work: Wang ZY; Data collection: Dai XY, Li PL, Ma JK, Ma YY, Tang YJ, Tian XQ, Song C; Supervision: Wang ZY; Analysis and interpretation of the data: Dai XY, Li PL, Ma JK, Ma YY, Tang YJ, Tian XQ; Statistical analysis: Wang ZY, Song C; Drafting the manuscript: Wang ZY; Critical revision of the manuscript: all authors; Approval of the final manuscript: all authors.

Funding

Joint Construction Project of Henan Provincial Medical Science and Technology Research Program (LHGJ20230524).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was conducted in accordance with the Declaration of Helsinki and approved by the Medical Research Ethics Committee of the First Affiliated Hospital of Henan Medical University (Approval Number: EC-LW026-051). Written informed consent was obtained from all parents/local guardians.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.