Hemophagocytic Lymphohistiocytosis Susceptibility (HLH) Factors IL-18 and Cytotoxic Impairment Converge on Pathologic CD8 T-cell Hyperactivation

Abstract

Excess IL-18 and graded defects in cytotoxicity are fundamental susceptibility factors for HLH and MAS. In mice, neither excess IL-18 (18tg mice) nor perforin-deficiency individually cause immunopathology without inflammatory challenge. However, 18tg mice lacking perforin (18tg; Prf1-/-) develop spontaneous lethal hyperinflammation, while subclinical MAS was found in 18tg;Prf1+/- mice. HLH and MAS patients demonstrate CD8 T-cell activation in tissue & peripheral blood. We sought to understand how IL-18 and cytotoxic impairment may interact in driving spontaneous hyperinflammation.We examined mice bearing single- and dual-susceptibility factors for “clinical” features of hyperinflammation, cytokine levels, and flow cytometric, transcriptional, functional, and TCR-seq phenotypes.Flow cytometric analysis of dual susceptibility mice showed spontaneous expansion of CD8 T-cells (and not CD4 T- or NK-cells), which expressed the IL-18 receptor and multiple inhibitory receptors (PD-1, Lag-3, CD39), yet overproduced IFNg. Such “hyperinflammatory CD8 T-cells” were present in reticuloendothelial organs (spleen, bone marrow, and liver) but not lymph nodes, peripheral blood, or thymus. Transcriptional analysis demonstrated hyperactivated CD8 T cells showed features of both terminal effector function and exhaustion when compared to well-described datasets. This analysis also revealed a strong signature for recent antigen activation. BulkCD8 TCR sequencing showed dramatic oligoclonal hyper-expansion of TCR sequences from dual susceptibility mice with no significant clonal overlap between biologic replicate samples. In vitro, TCR stimulation was necessary for IL-18 to promote IFNg production and restimulation-induced cell death (RICD), whereas perforin-deficiency prevented RICD. Broad-spectrum antibiotics did not rescue spontaneous hyperinflammation in dual-susceptibility mice, suggesting T-cell responses were not directed at commensal bacteria. Attempts to circumvent CD8 T-cell activation, using inducible CD8 T-cell-specific deletion of Il18r1 or by promoting allelic exclusion by fixing the T-cell receptor, showed breakthrough expansion of hyperactivated CD8 T-cells.Thus, IL-18 and cytotoxicity drive progressive, post-thymic, oligoclonal CD8 T-cell expansion that may underly life-threatening immunopathology in HLH and MAS. Expansion of hyperactivated CD8T cells in reticuloendothelial organs aligns with sites of hemophagocytosis. These data together implicate HLH/MAS susceptibility factors interact with TCR stimulation, whether by infectious triggers or as-yet undetermined self antigens, to drive potentially-targetable mechanisms of T-cell activation. [Display omitted] Supporting figures.