Among the caspases that cause regulated cell death, a unique function for caspase-7 has remained elusive. Caspase-3 performs apoptosis, whereas caspase-7 is typically considered an inefficient ...back-up. Caspase-1 activates gasdermin D pores to lyse the cell; however, caspase-1 also activates caspase-7 for unknown reasons
. Caspases can also trigger cell-type-specific death responses; for example, caspase-1 causes the extrusion of intestinal epithelial cell (IECs) in response to infection with Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium)
. Here we show in both organoids and mice that caspase-7-deficient IECs do not complete extrusion. Mechanistically, caspase-7 counteracts gasdermin D pores and preserves cell integrity by cleaving and activating acid sphingomyelinase (ASM), which thereby generates copious amounts of ceramide to enable enhanced membrane repair. This provides time to complete the process of IEC extrusion. In parallel, we also show that caspase-7 and ASM cleavage are required to clear Chromobacterium violaceum and Listeria monocytogenes after perforin-pore-mediated attack by natural killer cells or cytotoxic T lymphocytes, which normally causes apoptosis in infected hepatocytes. Therefore, caspase-7 is not a conventional executioner but instead is a death facilitator that delays pore-driven lysis so that more-specialized processes, such as extrusion or apoptosis, can be completed before cell death. Cells must put their affairs in order before they die.
Killer cell lectin-like receptor G1 (KLRG1) has traditionally been regarded as an inhibitory receptor of T cell exhaustion in chronic infection and inflammation. However, its exact role in hepatitis ...B virus (HBV) infection remains elusive. CD8+ T cells from 190 patients with chronic hepatitis B were analyzed ex vivo for checkpoint and apoptosis markers, transcription factors, cytokines and subtypes in 190 patients with chronic hepatitis B. KLRG1+ and KLRG1- CD8+ T cells were sorted for transcriptome analysis. The impact of the KLRG1-E-cadherin pathway on the suppression of HBV replication mediated by virus-specific T cells was validated in vitro. As expected, HBV-specific CD8+ T cells expressed higher levels of KLRG1 and showed an exhausted molecular phenotype and function. However, despite being enriched for the inhibitory molecules, thymocyte selection-associated high mobility group box protein (TOX), eomesodermin (EOMES), and Helios, CD8+ T cells expressing KLRG1 produced significant levels of tumour necrosis factor (TNF)-α, interferon (IFN)-γ, perforin, and granzyme B, demonstrating not exhausted but active function. Consistent with the in vitro phenotypic assay results, RNA sequencing (RNA-seq) data showed that signature effector T cell and exhausted T cell genes were enriched in KLRG1+ CD8+ T cells. Furthermore, in vitro testing confirmed that KLRG1-E-cadherin binding inhibits the antiviral efficacy of HBV-specific CD8+ T cells. Based on these findings, we concluded that KLRG1+ CD8+ T cells are not only a terminally exhausted subgroup but also exhibit functional diversity, despite inhibitory signs in HBV infection.
Cytotoxic lymphocytes fight pathogens and cancer by forming immune synapses with infected or transformed target cells and then secreting cytotoxic perforin and granzyme into the synaptic space, with ...potent and specific killing achieved by this focused delivery. The mechanisms that establish the precise location of secretory events, however, remain poorly understood. Here we use single cell biophysical measurements, micropatterning, and functional assays to demonstrate that localized mechanotransduction helps define the position of secretory events within the synapse. Ligand-bound integrins, predominantly the α
β
isoform LFA-1, function as spatial cues to attract lytic granules containing perforin and granzyme and induce their fusion with the plasma membrane for content release. LFA-1 is subjected to pulling forces within secretory domains, and disruption of these forces via depletion of the adaptor molecule talin abrogates cytotoxicity. We thus conclude that lymphocytes employ an integrin-dependent mechanical checkpoint to enhance their cytotoxic power and fidelity.
Gamma delta (GD) T cells are an unconventional T cell type present in both the epidermis and the dermis of human skin. They are critical to regulating skin inflammation, wound healing, and ...anti-microbial defense. Similar to CD8+ cytotoxic T cells expressing an alpha beta (AB) TCR, GD T cells have cytolytic capabilities. They play an important role in elimination of cutaneous tumors and virally infected cells and have also been implicated in pathogenicity of several autoimmune diseases. T cell cytotoxicity is associated with the expression of the pore forming protein Perforin. Perforin is an innate immune protein containing a membrane attack complex perforin-like (MACPF) domain and functions by forming pores in the membranes of target cells, which allow granzymes and reactive oxygen species to enter the cells and destroy them. Perforin-2, encoded by the gene
, is a newly discovered member of this protein family that is critical for clearance of intracellular bacteria. Cutaneous GD T cells express both Perforin and Perforin-2, but many questions remain regarding the role that these proteins play in GD T cell mediated cytotoxicity against tumors and bacterial pathogens. Here, we review what is known about Perforin expression by skin GD T cells and the mechanisms that contribute to Perforin activation.
For a long time, ex vivo-expanded peripheral-blood-derived γδT cell (PBγδT)-based immunotherapy has been attractive, and clinical trials have been undertaken. However, the difficulty in expanding ...cytotoxic γδT cells to an adequate number has been a major limitation to the efficacy of treatment in most cases. We successfully re-generated γδT cells from γδT cell-derived human induced pluripotent stem cells (iPSCs). The iPSC-derived γδT cells (iγδTs) killed several cancer types in a major histocompatibility complex (MHC)-unrestricted manner. Single-cell RNA sequencing (scRNA-seq) revealed that the iγδTs were identical to a minor subset of PBγδTs. Compared with a major subset of PBγδTs, the iγδTs showed a distinctive gene expression pattern: lower CD2, CD5, and antigen-presenting genes; higher CD7, KIT, and natural killer (NK) cell markers. The iγδTs expressed granzyme B and perforin but not interferon gamma (IFNγ). Our data provide a new source for γδT cell-based immunotherapy without quantitative limitation.
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•We successfully re-generated γδT cells from γδT cell-derived human iPSCs•The re-generated γδT cells showed cytotoxicity against cancer cell lines•Distinctive signatures of the re-generated γδT cells were identified
Murai et al. successfully re-generated γδT cells from γδT cell-derived human iPS cells. Molecular signatures of the re-generated γδT cells (iγδTs) were identical to those of a minor subset of ex vivo-expanded peripheral-blood-derived γδT cells. Cytotoxicity of iγδTs against several cancer cell lines was exhibited in an MHC-unrestricted manner. The novel cell resource will advance off-the-shelf γδT cell-based immunotherapy.
Perforin secreted from cytotoxic lymphocytes plays a critical role in cancer immunosurveillance. The aim of this study was to investigate the therapeutic potential of liposomes containing perforin ...expression vector driven by the promotor of prostate-specific antigen (PSA). The anti-tumor effect of perforin was analyzed using prostate cancer (PC) PC-3 cells in which perforin expression was controlled by Tet-on system (PC-3PRF cells). Liposomes encapsulating PSA promoter-driven perforin expression vector (pLipo) were constructed for its specific expression in PC. The anti-tumor effect of pLipo was evaluated in vitro using docetaxel-resistant PC 22Rv1 PC cell line, 22Rv1DR, and PC-3 cells in the presence of human peripheral blood mono nuclear cells (PBMCs) and also in vivo using male nude mice bearing 22Rv1DR cell-derived tumor xenograft. Induction of perforin significantly inhibited growth of PC-3PRF cells. Treatment with pLipo induced perforin expression in 22Rv1DR cells expressing PSA but not in PC-3 cells lacking it. Treatment with pLipo at a low concentration was prone to inhibit growth of both cell lines and significantly inhibited growth of 22Rv1DR cells when co-incubated with PBMCs. The combined use of pLipo at a high concentration with PBMCs showed nearly complete inhibition of 22Rv1DR cell growth. Intravenous administration of pLipo via tail vein increased the level of perforin in tumor and serum and significantly decreased the tumor volume. Our results suggest that liposome-mediated PC-specific expression of perforin could be a novel therapy for advanced PC.
The structure-activity relationships for a series of arylsulphonamide-based inhibitors of the pore-forming protein perforin have been explored. Perforin is a key component of the human immune ...response, however inappropriate activity has also been implicated in certain auto-immune and therapy-induced conditions such as allograft rejection and graft versus host disease. Since perforin is expressed exclusively by cells of the immune system, inhibition of this protein would be a highly selective strategy for the immunosuppressive treatment of these disorders. Compounds from this series were demonstrated to be potent inhibitors of the lytic action of both isolated recombinant perforin and perforin secreted by natural killer cells in vitro. Several potent and soluble examples were assessed for in vivo pharmacokinetic properties and found to be suitable for progression to an in vivo model of transplant rejection.
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•A new series of arylsulphonamides that inhibit perforin have been identified.•Potency against both recombinant perforin and whole NK cells was optimised.•In vitro toxicity, solubility and stability was measured for selected candidates.•In vivo PK was determined to select potential candidates for efficacy studies.•Several compounds were selected for evaluation in a mouse transplant rejection model.
Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome, characterized by severe hyperinflammation and immunopathological manifestations in several tissues. These features result from ...organ infiltration by overactivated CD8 T-cells and macrophages, which produce high levels of pro-inflammatory cytokines, such as IFN-γ, TNF-α, IL-6, and IL-18. Recently, several Janus kinase 1/2 (JAK1/2) inhibitors, such as ruxolitinib, have been developed as immunosuppressive agents. They have proven beneficial effects in the treatment of myeloproliferative disorders and inflammatory conditions. To determine whether pharmacological inhibition of the JAK1/2 not only prevents the onset of HLH immunopathology but also is effective against existing HLH, cytotoxicity-impaired Prf1−/− and Rab27a−/− mice with full-blown HLH syndrome were treated with a clinically relevant dose of ruxolitinib. In vivo, ruxolitinib treatment suppressed signal transducer and activator of transcription 1 activation and led to recovery from HLH manifestations in both murine models. In the Prf1−/− mice, these beneficial effects were evidenced by a greater survival rate, and in both murine models, they were evidenced by the correction of blood cytopenia and a rapid decrease in serum IL-6 and TNF-α levels. During ruxolitinib treatment, liver tissue damage receded concomitantly with a decrease in the number of infiltrating inflammatory macrophages and an increase in the number of alternatively activated macrophages. In Rab27a−/− mice, central nervous system involvement was significantly reduced by ruxolitinib therapy. Our findings demonstrate that clinically relevant doses of the JAK1/2 inhibitor ruxolitinib suppresses the harmful consequences of macrophage overactivation characterizing HLH in 2 murine models. The results could be readily translated into the clinic for the treatment of primary, and perhaps even secondary, forms of HLH.
•Treatment with clinical dose of JAK1/2 inhibitor (ruxolitinib) countered manifestations of HLH in 2 cytotoxicity-impaired murine models.•JAK1/2 inhibitor therapy in mice is effective on survival, cytopenia, inflammatory syndrome, central nervous system involvement, and liver tissue repair.
Natural Killer (NK) cells can engage multiple virally infected or tumor cells sequentially and deliver perforin for cytolytic killing of these targets. Using microscopy to visualize degranulation ...from individual NK cells, we found that repeated activation via the Fc receptor CD16 decreased the amount of perforin secreted. However, perforin secretion was restored upon subsequent activation via a different activating receptor, NKG2D. Repeated stimulation via NKG2D also decreased perforin secretion, but this was not rescued by stimulation via CD16. These different outcomes of sequential stimulation could be accounted for by shedding of CD16 being triggered by cellular activation. The use of pharmacological inhibitors and NK cells transfected to express a noncleavable form of CD16 revealed that CD16 shedding also increased NK cell motility and facilitated detachment of NK cells from target cells. Disassembly of the immune synapse caused by CD16 shedding aided NK cell survival and boosted serial engagement of target cells. Thus, counterintuitively, shedding of CD16 may positively impact immune responses.
Decidual natural killer （dNK） cells actively participate in the establishment and maintenance of maternal-fetal immune tolerance and act as local guardians against infection. However, how dNK cells ...maintain the immune balance between tolerance and anti-infection immune responses during pregnancy remains unknown. Here, we demonstrated that the inhibitory molecule T-cell immunoglobulin domain and mucin domain-containing molecule-3 （Tim-3） are expressed on over 60% of dNK cells. Tim-3^＋ dNK cells display higher interleukin （IL）-4 and lower tumor necrosis factor （TNF）-α and perforin production. Human trophoblast cells can induce the transformation of peripheral NK cells into a dNK-like phenotype via the secretion of galectin-9 （Gal-9） and the interaction between Gal-9 and Tim-3. In addition, trophoblasts inhibit lipopolysaccharide （LPS）-induced pro-inflammatory cytokine and perforin production by dNK cells, which can be attenuated by Tim-3 neutralizing antibodies. Interestingly, a decreased percentage of Tim-3-expressing dNK cells were observed in human miscarriages and murine abortion-prone models. Moreover, T helper （Th）2-type cytokines were decreased and Thl-type cytokines were increased in Tim-3^＋ but not Tim-3- dNK cells from human and mouse miscarriages. Therefore, our results suggest that the Gal-9/Tim-3 signal is important for the regulation of dNK cell function, which is beneficial for the maintenance of a normal pregnancy.
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