Modeling the HLH immune synapse uncovers critical roles for IS termination, cytokine intensity, and target cell death Available to Purchase

• Impaired IS termination and enhanced CTL cytokine production intensity were distinct and synergistic HLH-driving mechanisms.

• An IL-18/IFN-γ axis promoted RIPK1-dependent necroptotic cell death and HLH immunopathology, offering a new potential therapeutic target.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening systemic hyperinflammatory syndrome arising in many contexts. Its underlying mechanisms are often unclear, but defective granule-mediated cytotoxicity (familial HLH) and excess interleukin-18 (IL-18; macrophage activation syndrome) provide clues. Mounting evidence suggests the causes of HLH converge on cytotoxic T lymphocyte (CTL) hyperactivation and interferon gamma (IFN-γ) overproduction. We refined an in vitro system to simultaneously quantify multiple parameters of the murine CTL immune synapse (IS). Even in haploinsufficiency, perforin deficiency prolonged IS duration and increased IFN-γ/tumor necrosis factor production. Similarly, both target cell immortalization and inhibition of apoptotic caspases impaired IS termination and increased cytokine production. Strong CTL activation, through T-cell receptor or IL-18 signaling, also increased IFN-γ secretion but accelerated target cell death. Impaired IS termination synergized with strong CTL activation in driving IFN-γ production. Visually, both typical and Prf1^–/– CTL-IS terminated with apoptotic contraction. Serendipitously, we observed many IL-18–exposed CTL-IS terminated by target cell ballooning. Both IL-18–activated CTL and IFN-γ pretreatment caused up to half of target cells to die by receptor-interacting protein kinase 1 (RIPK1)–dependent necroptosis. In vivo, RIPK1 inhibition ameliorated virus-triggered HLH in Il18tg more than Prf1^–/– mice. By quantifying CTL-IS duration, cytokine production, and mode of cell death, we modeled multiple HLH contributors and their interactions and identified 3 HLH mechanistic categories: impaired IS termination, intense CTL cytokine production, and inflammatory target cell death. Integrating the inputs and outcomes of a hyperinflammatory CTL-IS may provide a useful framework for understanding, predicting, or treating HLH in its many forms.