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Niemann-Pick disease type C1 (NP-C1) is a rare lysosomal storage disorder resulting from mutations in an endolysosomal cholesterol transporter, NPC1. Despite typically presenting with pronounced neurological manifestations, NP-C1 also resembles long-term congenital immunodeficiencies that arise from impairment of cytotoxic T lymphocyte (CTL) effector function. CTLs kill their targets through exocytosis of the contents of lysosome-like secretory cytotoxic granules (CGs) that store and ultimately release the essential pore-forming protein perforin and proapoptotic serine proteases, granzymes, into the synapse formed between the CTL and target cell. We discovered that NPC1 deficiency increases CG lipid burden, impairs autophagic flux through stalled trafficking of the transcription factor EB (TFEB), and dramatically reduces CTL cytotoxicity. Using a variety of immunological and cell biological techniques, we found that the cytotoxic defect arises specifically from impaired perforin pore formation. We demonstrated defects of CTL function of varying severity in patients with NP-C1, with the greatest losses of function associated with the most florid and/or earliest disease presentations. Remarkably, perforin function and CTL cytotoxicity were restored in vitro by promoting lipid clearance with therapeutic 2-hydroxypropyl-β-cyclodextrin; however, restoration of autophagy through TFEB overexpression was ineffective. Overall, our study revealed that NPC1 deficiency has a deleterious impact on CTL (but not natural killer cell) cytotoxicity that, in the long term, may predispose patients with NP-C1 to atypical infections and impaired immune surveillance more generally.

Familial forms of the severe immunoregulatory disease Haemophagocytic Lymphohistiocytosis (HLH) arise from bi-allelic mutations in the PRF1, UNC13D, STXBP2 and STX11 genes. Early and accurate diagnosis of the disease is important to determine the most appropriate treatment option, including potentially curative stem cell transplantation. The diagnosis of familial HLH (FHL) is traditionally based on finding bi-allelic mutations in patients with HLH symptoms and reduced natural killer (NK) cell cytotoxicity. However, patients often have a low NK cell count or receive immunosuppressive therapies that may render the NK cytotoxicity assay unreliable. Furthermore, to fully understand the nature of a disease it is critical to directly assess the effect of mutations on cellular function; this will help to avoid instances where carriers of innocuous mutations may be recommended for invasive procedures including transplantation. To overcome this diagnostic problem, we have developed a rapid and robust method that takes advantage of the functional equivalence of the human and mouse orthologues of PRF1, UNC13D, STX11 and STXBP2 proteins. By knocking out endogenous mouse genes in CD8+ T cells and simultaneously replacing them with their mutated human orthologues, we can accurately assess the effect of mutations on cell function. The wide dynamic range of this novel system allowed us to understand the basis of otherwise cryptic cases of FHL/HLH and, in some instances, to demonstrate that previously reported mutations are unlikely to cause FHL. This novel approach provides valuable new information to enable more accurate diagnosis and treatment of HLH/FHL patients who inherit mutations of undetermined pathogenicity.

Perforin is a pore forming protein used by cytotoxic T lymphocytes to remove cancerous or virus-infected cells during the immune response. During the response, the lymphocyte membrane becomes refractory to perforin function by accumulating densely ordered lipid rafts and externalizing negatively charged lipid species. The dense membrane packing lowers the capacity of perforin to bind, and the negatively charged lipids scavenge any residual protein before pore formation. Using atomic force microscopy on model membrane systems, we here provide insight into the molecular basis of perforin lipid specificity., Physical membrane properties play a determining role in defining the sensitivity of membranes to the immune effector perforin.

Killer T cells (cytotoxic T lymphocytes, CTLs) maintain immune homoeostasis by eliminating virus-infected and cancerous cells. CTLs achieve this by forming an immunological synapse with their targets and secreting a pore-forming protein (perforin) and pro-apoptotic serine proteases (granzymes) into the synaptic cleft. Although the CTL and the target cell are both exposed to perforin within the synapse, only the target cell membrane is disrupted, while the CTL is invariably spared. How CTLs escape unscathed remains a mystery. Here, we report that CTLs achieve this via two protective properties of their plasma membrane within the synapse: high lipid order repels perforin and, in addition, exposed phosphatidylserine sequesters and inactivates perforin. The resulting resistance of CTLs to perforin explains their ability to kill target cells in rapid succession and to survive these encounters. Furthermore, these mechanisms imply an unsuspected role for plasma membrane organization in protecting cells from immune attack., Cytotoxic T lymphocytes (CTLs) eliminate virus-infected and cancerous cells by secreting the pore-forming protein (perforin) and pro-apoptotic serine proteases (granzymes). Here authors show that two mechanisms protect the membranes of CTLs from disruption by perforin and granzymes.

A series of novel 5-arylidene-2-thioxoimidazolidin-4-ones were investigated as inhibitors of the lymphocyte-expressed pore-forming protein perforin. Structure-activity relationships were explored through variation of an isoindolinone or 3,4-dihydroisoquinolinone subunit on a fixed 2-thioxoimidazolidin-4-one/thiophene core. The ability of the resulting compounds to inhibit the lytic activity of both isolated perforin protein and perforin delivered in situ by natural killer cells was determined. A number of compounds showed excellent activity at concentrations that were nontoxic to the killer cells, and several were a significant improvement on previous classes of inhibitors, being substantially more potent and soluble. Representative examples showed rapid and reversible binding to immobilized mouse perforin at low concentrations (≤2.5 μM) by surface plasmon resonance and prevented formation of perforin pores in target cells despite effective target cell engagement, as determined by calcium influx studies. Mouse PK studies of two analogues showed T1/2 values of 1.1-1.2 h (dose of 5 mg/kg i.v.) and MTDs of 60-80 mg/kg (i.p.).

Jenkins et al. discover that failure of perforin and granzyme cytotoxicity by human and mouse CTLs/NK cells prolongs the immunological synapse, leading to repetitive calcium signaling and hypersecretion of inflammatory mediators that subsequently activate macrophages. Disengagement from target cells is dependent on apoptotic caspase signaling. The findings may provide mechanistic understanding for immunopathology in familial hemophagocytic lymphohistiocytosis., Failure of cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells to kill target cells by perforin (Prf)/granzyme (Gzm)-induced apoptosis causes severe immune dysregulation. In familial hemophagocytic lymphohistiocytosis, Prf-deficient infants suffer a fatal “cytokine storm” resulting from macrophage overactivation, but the link to failed target cell death is not understood. We show that prolonged target cell survival greatly amplifies the quanta of inflammatory cytokines secreted by CTLs/NK cells and that interferon-γ (IFN-γ) directly invokes the activation and secondary overproduction of proinflammatory IL-6 from naive macrophages. Furthermore, using live cell microscopy to visualize hundreds of synapses formed between wild-type, Prf-null, or GzmA/B-null CTLs/NK cells and their targets in real time, we show that hypersecretion of IL-2, TNF, IFN-γ, and various chemokines is linked to failed disengagement of Prf- or Gzm-deficient lymphocytes from their targets, with mean synapse time increased fivefold, from ∼8 to >40 min. Surprisingly, the signal for detachment arose from the dying target cell and was caspase dependent, as delaying target cell death with various forms of caspase blockade also prevented their disengagement from fully competent CTLs/NK cells and caused cytokine hypersecretion. Our findings provide the cellular mechanism through which failed killing by lymphocytes causes systemic inflammation involving recruitment and activation of myeloid cells.

The effective engagement of cytotoxic lymphocytes (CLs) with their target cells is essential for the removal of virus-infected and malignant cells from the body. The spatiotemporal properties that define CL engagement and killing of target cells remain largely uncharacterized due to a lack of biological reporters. We have used a novel live cell microscopy technique to visualize the engagement of primary human and mouse CL with their targets and the subsequent delivery of the lethal hit. Extensive quantitative real-time analysis of individual effector–target cell conjugates demonstrated that a single effector calcium flux event was sufficient for the degranulation of human CLs, resulting in the breach of the target cell membrane by perforin within 65–100 s. In contrast, mouse CLs demonstrated distinct calcium signaling profiles leading to degranulation: whereas mouse NKs required a single calcium flux event, CD8+ T cells typically required several calcium flux events before perforin delivery. Irrespective of their signaling profile, every target cell that was damaged by perforin died by apoptosis. To our knowledge, we demonstrate for the first time that perforin pore delivery is unidirectional, occurring exclusively on the target cell membrane, but sparing the killer cell. Despite this, the CTL membrane was not intrinsically perforin resistant, as intact CTLs presented as targets to effector CTLs were capable of being killed by perforin-dependent mechanisms. Our results highlight the remarkable efficiency and specificity of perforin pore delivery by CLs.