Single-cell immunophenotyping reveals mutation-specific immune alterations in severe congenital neutropenia

Abstract Introduction: Congenital defects in neutrophil number or function are associated with severe, recurrent infections often requiring intensive medical care. While pyogenic infectious complications in inherited neutrophil disorders are well recognized, the degree of adaptive and innate immunodeficiency as well as immune dysregulation remain poorly described. In this study, we performed single-cell immunophenotyping to characterize cellular immune profiles in the most common forms of severe congenital neutropenia (SCN). Methods: Cell subpopulations were analyzed in peripheral blood samples using cytometry by time of flight (CyTOF). Immunophenotyping employed the Maxpar Direct Immune Profiling Assay, using a 34-marker panel, which enabled identification of 39 white blood cell subpopulations via Cytobank software. A machine learning algorithm was applied to classify cells based on a control reference using R software. High-dimensional data were visualized with the tSNE-CUDA algorithm. Cell subset proportions were compared between SCN subtypes, Glycogen Storage Disease Type 1b (GSD1b), immunological neutropenia and control group using the one-way ANOVA with Tukey's multiple comparisons test or the Kruskal-Wallis test with Dunn's post hoc test, depending on data distribution. Results: A total of 36 patients were enrolled: 27 with SCN, 4 with immunological neutropenia, and 5 healthy donors (aged 1-46 years; female/male ratio 17/19). SCN patients carried mutations in the following genes: ELANE (n=7), CLPB (n=7), SLC37A4 (GSD1b; n=7), CXCR2 (n=2), SRP54 (n=2), and TCIRG1 (n=2). Compared to controls, patients with SCN had a significantly higher proportion of classical monocytes (10.6% vs 4.4%; p=0.0249). ELANE-mutated patients showed elevated classical monocytes compared to CLPB (24.3% vs 8.4%; p=0.0017), GSD1b (4.9%; p=0.0005), and immunological neutropenia (6.7%; p=0.0188). Basophils were increased in ELANE patients relative to controls (1.4% vs 0.6%; p=0.05), GSD1b (0.5%; p=0.0018), and immunological neutropenia (0.5%; p=0.0161). CLPB-mutated patients had higher plasmacytoid dendritic cells (pDC) than GSD1b (0.4% vs 0.1%; p=0.0228). Apart from expected variation in the myeloid compartment there are also disease related cellular traits related to adaptive and innate immunity. Specifically, GSD1b patients exhibited reduced CD8+ naïve T cells compared to controls (8.8% vs 19.8%; p=0.0073) and SCN (14.7%; p=0.0339), particularly ELANE (19.6%; p=0.0097). CD4+ naïve T cells were lower in GSD1b compared to controls (24.0% vs 39.4%; p=0.0065) and immunological neutropenia (43.0%; p=0.0076). SCN patients had reduced CD4+ naïve T cells compared to controls (24.0% vs 39.4%; p=0.0214) and immunological neutropenia (43.0%; p=0.0250), driven mainly by ELANE (19.9%; p=0.0338, p=0.0367 respectively). The CD4- MAIT/NKT cell subset was significantly elevated in GSD1b compared to controls (10.8% vs 1.4%; p= 0.0006) and immunological neutropenia (1.5%; p= 0.0039). GSD1b patients also exhibited markedly reduced memory regulatory T cells (Treg memory) compared to controls (0.3% vs 2.2%; p<0.0001), immunological neutropenia (2.1%; p=0.0072) and SCN (1.8%; p=0.0011), particularly driven by CLPB (1.9%; p=0.0082 respectively). Conclusions: This single-cell immunophenotyping study reveals distinct immune signatures among SCN subtypes. ELANE-mutated patients displayed elevated classical monocytes and basophils, suggesting heightened innate immune activation. In contrast, GSD1b patients showed profound T cell dysregulation, including reduced naïve CD4⁺ and CD8⁺ T cells, expansion of CD4⁻ MAIT/NKT cells, and a marked deficit in memory regulatory T cells, indicating impaired immune tolerance and metabolic-immune crosstalk. CLPB-related SCN was associated with increased plasmacytoid dendritic cells. These findings highlight distinct immunological signatures among SCN subtypes, with potential implications for understanding non-pyogenic immune complications in neutropenia. Funding: The project was financed by National Science Centre, Poland, PRELUDIUM 2023/49/N/NZ6/02818 and NAWA – Polish National Agency for Academic Exchange in cooperation with Medical Research Agency under the Walczak Programme, BPN/WAL/2023/1/00008 and by the Foundation for Polish Science TEAM NET Program, POIR.04.04.00-00-1603/18.