<scp>CD8</scp> ⁺ T cells surpass <scp>NK</scp> cells in granule release assay: Implications for diagnosing primary <scp>HLH</scp>

Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome caused by impaired cytotoxic function of natural killer (NK) cells and CD8+ T lymphocytes.1 In primary HLH, inherited defects in genes regulating granule-mediated cytotoxicity including PRF1, UNC13D, STX11, STXBP2, RAB27A, AP3B1 and LYST result in failure to eliminate activated immune cells, leading to uncontrolled cytokine release and progressive organ dysfunction.2-6 Early recognition is essential but challenging, as clinical manifestations are nonspecific and frequently mimic infection, malignancy or autoimmune disease.7 Although established diagnostic criteria provide a structured framework,1 genetic confirmation is often not immediately available in urgent clinical settings. Functional evaluation of lymphocyte degranulation, commonly assessed by CD107a lysosomal-associated membrane protein 1 (LAMP-1) surface expression following stimulation, therefore serves as an important adjunct in the diagnostic pathway.8 However, conventional assays typically require peripheral blood mononuclear cell (PBMC) isolation, target cell stimulation or specialized infrastructure, limiting rapid and widespread implementation.9 We previously reported a simplified whole-blood, phorbol 12-myristate 13-acetate (PMA)/ionophore-based NK-cell degranulation assay as a practical screening tool.10 In the present study, we extend this approach to enable simultaneous assessment of CD8+T-cell degranulation and systematically compare the diagnostic performance of NK and CD8+ T-cell compartments in primary HLH. The study was approved by the Institutional Ethics Committee of Indian Council Of Medical Research (ICMR)-National Institute of Immunohematology and conducted in accordance with the Declaration of Helsinki. Sixty participants were enrolled, including 21 patients with genetically confirmed primary HLH and 7 patients with secondary HLH or other hyperinflammatory syndromes. Twenty-seven healthy controls aged 20–40 years were recruited to minimize variability related to immune maturation and age-associated immune senescence. In addition, five paediatric healthy controls aged <1 year were included. Primary HLH genotypes included: familial haemophagocytic lymphohistiocytosis type 2 (FHL2) (PRF1, n = 8), FHL3 (UNC13D, n = 7), FHL4 (STX11, n = 1), FHL5 (STXBP2, n = 2), Griscelli syndrome type 2 (RAB27A, n = 1), Hermansky–Pudlak syndrome type 2 (AP3B1, n = 1) and Chediak–Higashi syndrome (LYST, n = 1). Genetic variants were classified according to the criteria of the American College of Medical Genetics and Genomics (ACMG) as pathogenic or likely pathogenic. White blood cell counts at the time of sampling and any prior exposure to lymphotoxic or immunosuppressive therapy were recorded. Samples obtained shortly after lymphotoxic therapy were interpreted with caution, as therapy-related cytopenias could affect assay performance and potentially lead to misleading results. All these data are summarized in Table S1. One hundred microlitres of heparin-anticoagulated whole blood (absolute lymphocyte count approximately 1 × 103/μL) was incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD107a antibody and stimulated with PMA (0.15 μg/mL) plus calcium ionophore (3 μg/mL) in Roswell Park Memorial Institute medium (RPMI) (final volume 500 μL). Incubation was performed for 2 h at 37°C in 5% CO2. Following stimulation, surface staining was performed using CD56-PC7, Anti-CD3 antibody conjugated with R-Phycoerythrin (CD3-PE) and Allophycocyanin-Alexa Fluor 750 (CD8-APC-A750). Red blood cell lysis was completed prior to acquisition. This protocol permits simultaneous evaluation of NK and CD8+ T cells within the same 100-μL whole-blood tube, avoiding the need for additional sample volume. Lymphocytes were identified using forward- and side-scatter characteristics. Doublets were excluded using forward scatter area (FSC-A) versus forward scatter height (FSC-H) gating. CD107a expression was analysed within NK cells (CD56+CD3−) and CD8+T cells (CD3+CD8). Samples with extremely low NK-cell counts were excluded from NK analysis but retained for CD8+ T-cell evaluation. CD107a expression was quantified as percentage of positive cells. Cut-offs for abnormal expression were defined using unstimulated controls and healthy control distributions. For analysis, at least 100 CD56+ NK-cell events were acquired within the lymphocyte gate to ensure reliable evaluation of NK-cell degranulation. Data are presented as median (interquartile range, IQR). Group comparisons were performed using the Mann–Whitney U-test. Sensitivity and specificity were calculated using receiver operating characteristic analysis (ROC). A value of p < 0.05 was considered statistically significant. Analyses were performed using Graph Pad Prism (Graph Pad Software, San Diego, CA, USA). CD107a expression on NK cells and CD8+ T cells is summarized in Table 1. Healthy controls demonstrated robust CD107a upregulation following stimulation, with significantly higher median CD107a expression on CD8+ T cells compared with NK cells (paired p < 0.0001). Patients with defects in granule trafficking or exocytosis (FHL3–5) showed markedly reduced CD107a expression on both NK cells (median—2.7%) and CD8+ T cells (median—6.7%), consistent with impaired degranulation. In contrast, patients with FHL2 exhibited preserved CD107a expression on both cell types, reflecting intact degranulation despite defective cytotoxicity. Patients with secondary HLH demonstrated increased CD107a expression after stimulation compared with unstimulated cells in both NK and CD8+ T cells. All 55 evaluable samples are summarized graphically Figure S1, including individual data points grouped by genotype and clinical category. A representative flow cytometry plot showing the gating strategy and CD107a expression on NK cells and CD8+ T cells in healthy controls, secondary HLH and different HLH subtypes are presented in Figure 1. Using ROC-derived thresholds, impaired degranulation was defined as: <46.4% for CD8+T cells and <20.6% for NK cells. Applying these cut-offs, CD8+T-cell degranulation demonstrated a sensitivity of 100% and specificity of 96.3%, whereas NK-cell degranulation showed a sensitivity of 92.3% and specificity of 85.2%. These findings indicate superior diagnostic performance of CD8+ T-cell degranulation compared with NK-cell analysis in this cohort. In two patients with severe cytopenia, NK-cell analysis was not feasible due to extremely low cell counts (≤100/μL); however, CD8+ T-cell degranulation remained evaluable and was markedly reduced. One patient with a UNC13D mutation (p.Leu940ArgfsTer13) demonstrated preserved CD107a expression on NK cells but reduced expression on CD8+ T cells. Conversely, one patient with Griscelli syndrome type 2 (RAB27A) showed borderline CD107a expression on NK cells (20%) but reduced expression on CD8+ T cells (42%) compared with healthy controls. HLH remains diagnostically challenging due to its heterogeneous presentation and overlap between primary and secondary forms.7 Rapid diagnosis is critical, as delayed recognition of primary cytotoxic pathway defects can result in rapid clinical deterioration and high mortality.11 Although next-generation sequencing facilitates molecular diagnosis, it is time-consuming, may miss non-coding variants and frequently identifies variants of uncertain significance requiring functional validation.12 In contrast, flow cytometry-based assessment of perforin expression and granule release assays provides rapid functional diagnosis, facilitates early identification of underlying cytotoxic pathway defects and remains central to guiding genetic evaluation and clinical decision-making.9 Conventional determination of CD107a expression on NK cells is typically performed using PBMCs or purified NK cells stimulated with the K562 target cell line.8 However, K562-based assays require dedicated cell culture facilities and may introduce interlaboratory variability. Only a limited number of studies have reported simultaneous evaluation of NK- and CD8+ T-cell degranulation in HLH. Bryceson et al. demonstrated high sensitivity and specificity using combined T-cell receptor (TCR)-triggered CD8+ T-cell assays and Fc receptor-triggered NK-cell assays; however, this approach requires prior cell separation, larger blood volumes, defined stimulants and technical expertise.13 In contrast, our whole-blood PMA/ionophore-based CD107a assay enables parallel assessment of NK-cell and CD8+ T-cell degranulation using minimal sample volume and simplified processing while maintaining comparable sensitivity and specificity. As described by Byson et al., the diagnostic performance of our assay is further enhanced by incorporating CD57 expression within the CD8+ T-cell compartment, enabling more precise characterization of terminally differentiated cytotoxic effector subsets.13 This streamlined strategy is particularly advantageous in paediatric and cytopenic patients and in settings where rapid turnaround time is essential. Simultaneous stimulation of both cytotoxic compartments increases diagnostic confidence and reduces inconclusive results due to low cell counts or borderline responses. Consistent with prior reports,2-6 patients with granule trafficking and exocytosis defects (FHL3–5, CHS) showed markedly reduced CD107a expression in both NK and CD8+ T cells, whereas preserved CD107a upregulation was observed in perforin deficiency (PRF1), reflecting intact degranulation despite impaired cytotoxicity. Simultaneous assessment enhanced diagnostic accuracy, with CD8+ T-cell exocytosis more consistently identifying defects in UNC13D, STXBP2, STX11 and RAB27A. In selected cases, preserved NK-cell CD107a expression with reduced CD8+ T-cell degranulation suggested greater sensitivity of the CD8+T-cell compartment for detecting partial or hypomorphic defects. Importantly, CD8+ T-cell analysis remained feasible in samples with severe NK-cell lymphopenia. Interpretation of exocytosis assays may be influenced by lymphopenia, infection, systemic inflammation, prior therapy and interlaboratory variability. Laboratory-specific cut-offs and careful clinical correlation are therefore essential.9 Multicentre validation of simplified whole-blood protocols will be important to standardize thresholds and enhance reproducibility. In summary, simultaneous evaluation of NK- and CD8+ T-cell degranulation strengthens functional screening for HLH. The simplified whole-blood CD107a assay is rapid, low volume and cost-effective, with diagnostic performance comparable to established methods. Notably, CD8+ T-cell assessment enhances detection of granule trafficking and exocytosis defects, while the whole-blood format ensures practicality and accessibility. We propose this approach as a robust first-line screening strategy, reserving more complex assays for confirmatory or equivocal cases. Maya Gupta: Methodology; investigation; formal analysis; writing – original draft. Umair Ahmed Bargir: Data curation. Shweta Shinde: Methodology; investigation; formal analysis. Manisha Madkaikar: Conceptualization; project administration; writing – review and editing. Reetika Malik Yadav: Data curation. Lavina Temkar: Formal analysis. Aparna Dalvi: Investigation. The data that supports the findings of this study are available in the supplementary material of this article. Figure S1. Table S1. 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