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/ IFNγ -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 IL-18 (Macrophage Activation Syndrome, MAS) provide clues. Mounting evidence suggests the causes of HLH converge on cytotoxic T lymphocyte (CTL) hyperactivation and overproduction of IFNγ. 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 IFNγ/TNF 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 IFNγ secretion but accelerated target cell death. Impaired IS termination synergized with strong CTL activation in driving IFNγ production. Visually, both typical and Prf1-/- CTL-IS terminated with apoptotic contraction. We serendipitously observed many IL-18 exposed CTL-IS terminated by target cell ballooning. Both IL-18-activated CTL and IFNγ pretreatment caused up to half of target cells to die by 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 three 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.