Introduction Hemophagocytic lymphohistiocytosis (HLH) is a severe inflammatory condition characterized by overt immune activation and cytokine storm. Without timely intervention, HLH rapidly progresses to multiple organ failure and almost certainly lead to death. HLH can be broadly categorized into primary and secondary HLH. While primary HLH (pHLH) is caused by genetic predisposition, secondary HLH (sHLH) can be triggered by a variety of factors, including infection, cancer and autoimmunity. Here we use single-cell expression and TCR profiling technologies to identify the trigger for aberrant activation of CD8+ cells in sHLH.

Methods Patients were diagnosed with NKTL according to the 2008 World Health Organization classification. Ten whole blood samples profiled for this study were from three patients with NKTCL and concomitant HLH, four patients with NKTCL without HLH and three healthy individuals without any hematological malignancies. All subjects in this study provided written informed consent.

Single cell 5' gene expression libraries, BCR and TCR libraries were prepared using the 10X Genomics Chromium Next GEM Single Cell 5′v2 Library & Gel Bead Kit and V(D)J Enrichment Kits according to the manufacturer's protocol.

Results Peripheral blood mononuclear cells (PBMCs) from sHLH patients, NKTL patients without sHLH, and healthy controls (HCs) were analyzed. sHLH patients displayed significantly elevated levels of activated CD8+ T cells and an inverted CD8:CD4 ratio, indicative of heightened proliferation and activation.

Single-cell RNA sequencing identified unique subsets of CD8+ T cells in sHLH patients, particularly CD38+Ki67+ effector memory T cells (Act-TEM), which were nearly absent in NKTL and HCs. These cells exhibited markers of exhaustion (PD-1, TIGIT, and EOMES) but retained active proliferation and cytotoxic capacity, diverging from canonical T-cell developmental trajectories. Gene set enrichment analysis highlighted their enrichment in interferon response and cell proliferation pathways, contrasting with the NFκB activation pathways observed in typical effector memory T cells (TEM).

T-cell receptor (TCR) sequencing also revealed no significant oligoclonal expansion in sHLH patients, suggesting a non-TCR-mediated activation mechanism. Naïve CD8+ T cells in sHLH patients produced granzyme B, further supporting non-canonical activation. Ex vivo cytokine treatments identified IL-15 as a key driver of this phenotype, capable of inducing granzyme B production and promoting the differentiation of CD8+ T cells into Act-TEM. Elevated IL-15 levels in plasma and monocytes, along with increased CCR5 expression on CD8+ T cells, corroborate the role of IL-15 in bystander activation too.

Conclusion We report IL-15 mediated bystanding activation of CD8+ T-cells in sHLH. This highlights IL-15 as a potential immune-modulating target to soothe the cytokine storm in patients with sHLH.

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2025
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