Acidic pH can attenuate immune killing through inactivation of perforin.

Cytotoxic lymphocytes are crucial to our immune system, primarily eliminating virus-infected or cancerous cells via perforin/granzyme killing. Perforin forms transmembrane pores in the plasma membrane, allowing granzymes to enter the target cell cytosol and trigger apoptosis. The prowess of cytotoxic lymphocytes to efficiently eradicate target cells has been widely harnessed in immunotherapies against haematological cancers.

Principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision.

Single-nucleotide variants (SNVs) are extremely prevalent in human cancers, although most of these remain clinically unactionable. The programmable RNA nuclease CRISPR-Cas13 has been deployed to specifically target oncogenic RNAs. However, silencing oncogenic SNVs with single-base precision remains extremely challenging due to the intrinsic mismatch tolerance of Cas13.

Cancer cell-specific PD-L1 expression is a predictor of poor outcome in patients with locally advanced oral cavity squamous cell carcinoma.

Background: Locally advanced oral cavity squamous cell carcinoma (OCSCC) presents a significant clinical challenge despite being partially responsive to standard treatment modalities. This study investigates the prognostic implications of programmed death-ligand 1 (PD-L1) expression in these tumors, focusing on its association with treatment outcomes and the immune microenvironment.

Methods: We assessed tumor-infiltrating lymphocytes (TILs) in 132 patients with OCSCC to evaluate their impact on survival.

Pharmacologic inhibition of dipeptidyl peptidase 1 (cathepsin C) does not block granzyme-mediated target cell killing by CD8 T or NK cells.

Recently developed small-molecule inhibitors of the lysosomal protease dipeptidyl peptidase 1 (DPP1), also known as cathepsin C (CatC), can suppress suppurative inflammation by blocking the processing of zymogenic (pro-) forms of neutrophil serine proteases (NSPs), including neutrophil elastase, proteinase 3, and cathepsin G. DPP1 also plays an important role in activating granzyme serine proteases that are expressed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Therefore, it is critical to determine whether DPP1 inhibition can also cause off-target suppression of CTL/NK-cell-mediated killing of virus-infected or malignant cells.

Single-base tiled screen unveils design principles of PspCas13b for potent and off-target-free RNA silencing.

The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized.

CRISPR-Cas13b-mediated suppression of HBV replication and protein expression.

Background & Aims: New antiviral approaches that target multiple aspects of the HBV replication cycle to improve rates of functional cure are urgently required. HBV RNA represents a novel therapeutic target. Here, we programmed CRISPR-Cas13b endonuclease to specifically target the HBV pregenomic RNA and viral mRNAs in a novel approach to reduce HBV replication and protein expression.

The CD8 T cell tolerance checkpoint triggers a distinct differentiation state defined by protein translation defects.

Peripheral CD8 T cell tolerance is a checkpoint in both autoimmune disease and anti-cancer immunity. Despite its importance, the relationship between tolerance-induced states and other CD8 T cell differentiation states remains unclear. Using flow cytometric phenotyping, single-cell RNA sequencing (scRNA-seq), and chromatin accessibility profiling, we demonstrated that in vivo peripheral tolerance to a self-antigen triggered a fundamentally distinct differentiation state separate from exhaustion, memory, and functional effector cells but analogous to cells defectively primed against tumors.

Fragment-based and structure-guided discovery of perforin inhibitors.

Perforin is a pore-forming protein whose normal function enables cytotoxic T and natural killer (NK) cells to kill virus-infected and transformed cells. Conversely, unwanted perforin activity can also result in auto-immune attack, graft rejection and aberrant responses to pathogens. Perforin is critical for the function of the granule exocytosis cell death pathway and is therefore a target for drug development.

Effects of a Small-Molecule Perforin Inhibitor in a Mouse Model of CD8 T Cell-Mediated Neuroinflammation.

Background And Objectives: Alteration of the blood-brain barrier (BBB) at the interface between blood and CNS parenchyma is prominent in most neuroinflammatory diseases. In several neurologic diseases, including cerebral malaria and Susac syndrome, a CD8 T cell-mediated targeting of endothelial cells of the BBB (BBB-ECs) has been implicated in pathogenesis.

Methods: In this study, we used an experimental mouse model to evaluate the ability of a small-molecule perforin inhibitor to prevent neuroinflammation resulting from cytotoxic CD8 T cell-mediated damage of BBB-ECs.

T-like CAR-T cells exhibit improved persistence and tumor control compared with conventional CAR-T cells in preclinical models.

Patients who receive chimeric antigen receptor (CAR)-T cells that are enriched in memory T cells exhibit better disease control as a result of increased expansion and persistence of the CAR-T cells. Human memory T cells include stem-like CD8 memory T cell progenitors that can become either functional stem-like T (T) cells or dysfunctional T progenitor exhausted (T) cells. To that end, we demonstrated that T cells were less abundant in infused CAR-T cell products in a phase 1 clinical trial testing Lewis Y-CAR-T cells (NCT03851146), and the infused CAR-T cells displayed poor persistence in patients.

A novel transcriptional signature identifies T-cell infiltration in high-risk paediatric cancer.

Background: Molecular profiling of the tumour immune microenvironment (TIME) has enabled the rational choice of immunotherapies in some adult cancers. In contrast, the TIME of paediatric cancers is relatively unexplored. We speculated that a more refined appreciation of the TIME in childhood cancers, rather than a reliance on commonly used biomarkers such as tumour mutation burden (TMB), neoantigen load and PD-L1 expression, is an essential prerequisite for improved immunotherapies in childhood solid cancers.

CD8 T Cells Promote Pathological Angiogenesis in Ocular Neovascular Disease.

Background: CD4 (cluster of differentation) and CD8 T cells are increased in the ocular fluids of patients with neovascular retinopathy, yet their role in the disease process is unknown.

Methods: We describe how CD8 T cells migrate into the retina and contribute to pathological angiogenesis by releasing cytokines and cytotoxic factors.

Results: In oxygen-induced retinopathy, flow cytometry revealed the numbers of CD4 and CD8 T cells were increased in blood, lymphoid organs, and retina throughout the development of neovascular retinopathy.

A cell-based functional assay that accurately links genotype to phenotype in familial HLH.

Familial forms of the severe immunoregulatory disease hemophagocytic lymphohistiocytosis (HLH) arise from biallelic 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 biallelic mutations in patients with HLH symptoms and reduced natural killer (NK)-cell cytotoxicity.

Beyond target cell death - Granzyme serine proteases in health and disease.

Granzymes are a family of small (∼32 kDa) serine proteases with a range of substrate specificities that are stored in, and released from, the cytoplasmic secretory vesicles ('granules') of cytotoxic T lymphocytes and natural killer cells. Granzymes are not digestive proteases but finely tuned processing enzymes that target their substrates in specific ways to activate various signalling pathways, or to inactivate viral proteins and other targets. Great emphasis has been placed on studying the pro-apoptotic functions of granzymes, which largely depend on their synergy with the pore-forming protein perforin, on which they rely for penetration into the target cell cytosol to access their substrates.

Small Molecule Inhibitors of Lymphocyte Perforin as Focused Immunosuppressants for Infection and Autoimmunity.

New drugs that precisely target the immune mechanisms critical for cytotoxic T lymphocyte (CTL) and natural killer (NK) cell driven pathologies are desperately needed. In this perspective, we explore the cytolytic protein perforin as a target for therapeutic intervention. Perforin plays an indispensable role in CTL/NK killing and controls a range of immune pathologies, while being encoded by a single copy gene with no redundancy of function.

Eprenetapopt triggers ferroptosis, inhibits NFS1 cysteine desulfurase, and synergizes with serine and glycine dietary restriction.

The mechanism of action of eprenetapopt (APR-246, PRIMA-1) as an anticancer agent remains unresolved, although the clinical development of eprenetapopt focuses on its reported mechanism of action as a mutant-p53 reactivator. Using unbiased approaches, this study demonstrates that eprenetapopt depletes cellular antioxidant glutathione levels by increasing its turnover, triggering a nonapoptotic, iron-dependent form of cell death known as ferroptosis. Deficiency in genes responsible for supplying cancer cells with the substrates for de novo glutathione synthesis (, , and ), as well as the enzymes required to synthesize glutathione ( and ), augments the activity of eprenetapopt.

The efficacy of combination treatment with elotuzumab and lenalidomide is dependent on crosstalk between natural killer cells, monocytes and myeloma cells.

Patients with refractory relapsed multiple myeloma respond to combination treatment with elotuzumab and lenalidomide. The mechanisms underlying this observation are not fully understood. Furthermore, biomarkers predictive of response have not been identified to date.

Preclinical Activity and Pharmacokinetic/Pharmacodynamic Relationship for a Series of Novel Benzenesulfonamide Perforin Inhibitors.

Perforin is a key effector of lymphocyte-mediated cell death pathways and contributes to transplant rejection of immunologically mismatched grafts. We have developed a novel series of benzenesulfonamide (BZS) inhibitors of perforin that can mitigate graft rejection during allogeneic bone marrow/stem cell transplantation. Eight such perforin inhibitors were tested for their murine pharmacokinetics, plasma protein binding, and their ability to block perforin-mediated lysis and to block the rejection of major histocompatibility complex (MHC)-mismatched mouse bone marrow cells.

IFN-γ cytotoxic CD4 T lymphocytes are involved in the pathogenesis of colitis induced by IL-23 and the food colorant Red 40.

The food colorant Red 40 is an environmental risk factor for colitis development in mice with increased expression of interleukin (IL)-23. This immune response is mediated by CD4 T cells, but mechanistic insights into how these CD4 T cells trigger and perpetuate colitis have remained elusive. Here, using single-cell transcriptomic analysis, we found that several CD4 T-cell subsets are present in the intestines of colitic mice, including an interferon (IFN)-γ-producing subset.

ESCRT-mediated membrane repair protects tumor-derived cells against T cell attack.

Cytotoxic T lymphocytes (CTLs) and natural killer cells kill virus-infected and tumor cells through the polarized release of perforin and granzymes. Perforin is a pore-forming toxin that creates a lesion in the plasma membrane of the target cell through which granzymes enter the cytosol and initiate apoptosis. Endosomal sorting complexes required for transport (ESCRT) proteins are involved in the repair of small membrane wounds.

Imaging immunity in patients with cancer using positron emission tomography.

Immune checkpoint inhibitors and related molecules can achieve tumour regression, and even prolonged survival, for a subset of cancer patients with an otherwise dire prognosis. However, it remains unclear why some patients respond to immunotherapy and others do not. PET imaging has the potential to characterise the spatial and temporal heterogeneity of both immunotherapy target molecules and the tumor immune microenvironment, suggesting a tantalising vision of personally-adapted immunomodulatory treatment regimens.

The pore conformation of lymphocyte perforin.

Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with proapoptotic granzymes, to bind to a target cell membrane where it oligomerizes and forms pores. The pores allow granzyme entry, which rapidly triggers the apoptotic death of the target cell.