Quantitation of Immune Synapse Duration Supports the Contribution of Perforin Haploinsufficiency to Hyperinflammation

Abstract

Abstract Hyperinflammation is an acute state that occurs in a variety of infectious, inflammatory, and genetic contexts. Familial Hemophagocytic Lymphohistiocytosis (FHL) is a rare Inborn Error of Immunity resulting from biallelic disruption of perforin or other genes necessary for granule-mediated cytotoxicity. Some reports suggest that heterozygous defects in FHL genes contribute to hyperinflammation in concert with infection or rheumatic diseases. Most known causes of hyperinflammation arise from abnormal regulation of the Immune Synapse (IS) between cytotoxic CD8 T-cells and target cells. Using live imaging, we sought to assess the effects of Perforin and other putative contributors to hyperinflammation on IS duration and magnitude. We evaluated the IS between CD8 T cells from mice with different T-cell receptor signal strengths (OT1 vs P14) and various target cell types, as well as that between Prf1+/+, +/−, and −/− P14 cells and BMDCs. IS duration was calculated based on time between killer cell calcium flux and target cell Propidium Iodide uptake, and cytokine ELISA of supernatants was performed. We found that OT1, but not P14, CD8 T-cells could kill MC57 fibrosarcoma cells, but both were able to kill BMDCs. Moreover, both complete Prf1 deficiency and Prf1 haploinsufficiency prolonged the IS and increased cytokine production. Excess IL18, a hyperinflammatory susceptibility factor that synergizes with Prf1 deficiency in vivo to drive spontaneous hyperinflammation, shortened IS duration but dramatically increased IFN-γ production. Thus, showing that our in vitro IS quantitation model is sensitive to small changes that may be important contributors to pathogenic T-cell activation and life-threating hyperinflammation. Supported by grants from NIH (R01 HD098428-03-04), Children's Hospital of Philadelphia (IDF FY21-FY24), and from Histiocytosis Association (FP00039723_A1)