Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening 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 important and complementary clues. Mounting evidence suggests the various genetic, infectious, malignancy-associated, and rheumatic causes of HLH all converge on cytotoxic T lymphocyte (CTL) hyperactivation and overproduction of IFNγ. Current clinical guidance emphasizes the need to address multiple HLH contributors but is challenging without a more functional mechanistic framework.
We developed an in vitro system to simultaneously quantify how multiple parameters of the murine CTL immune synapse (CTL-IS) responded to various HLH-related stimuli or specific cell death pathway inhibitors. We defined IS duration in CTL/target cell dyads as the time between CTL Ca++ flux and target cell PI uptake by live-cell microscopy. We also measured cytokine levels longitudinally in co-culture supernatants. We assessed the effects of cell death inhibition on HLH in vivo in Il18tg and Prf1-/- mice infected with LCMV(Armstrong).
Perforin haploinsufficiency prolonged IS duration and increased IFNγ production, demonstrating the system’s sensitivity. Target cell death resistance (immortalization or caspase inhibition) similarly prolonged CTL-IS duration and cytokine production, substantiating “Impaired IS Termination” as a category of HLH contributors. By contrast, strong CTL activation, via TCR or IL-18 signaling, increased IFNγ secretion but accelerated target cell death. This pattern, which we call “CTL Cytokine Production Intensity,” has been observed in CART IS and may represent a distinct category of HLH contributors. Surprisingly, IL-18 exposure drove some CTL-IS to terminate, even in the absence of perforin, via a morphologically inflammatory form of cell death inhibitable by blocking necroptosis. In vivo, RIPK1 inhibition ameliorated virus-triggered HLH in Il18tg but not Prfl-/- 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.
HLH contributors fall into three mechanistic categories: impaired IS termination, CTL cytokine production intensity, and inflammatory target cell death. (A) Still images from live cell microscopy of the CTL-IS. Green = Ca++ flux (fluo-4); red = target cell death (PI). (B) CTL-IS duration by Prf1 genotype with and without exogenous IL-18. (C) In vitro target cell death assay with IL-18, caspase-3/7 inhibition (Z-DEVD-FMK), and necroptosis inhibition (necrostatin-1). (D) Weight loss and plasma IFNg (Day 10) following LCMV Armstrong infection of the indicated mice with and without inhibition of necroptosis (Nec-1).
HLH contributors fall into three mechanistic categories: impaired IS termination, CTL cytokine production intensity, and inflammatory target cell death. (A) Still images from live cell microscopy of the CTL-IS. Green = Ca++ flux (fluo-4); red = target cell death (PI). (B) CTL-IS duration by Prf1 genotype with and without exogenous IL-18. (C) In vitro target cell death assay with IL-18, caspase-3/7 inhibition (Z-DEVD-FMK), and necroptosis inhibition (necrostatin-1). (D) Weight loss and plasma IFNg (Day 10) following LCMV Armstrong infection of the indicated mice with and without inhibition of necroptosis (Nec-1).
