Pharmacologic inhibition of dipeptidyl peptidase 1 (cathepsin C) does not block in vitro 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 in vivo 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. Herein, we demonstrate that the processing of human granzymes A and B, transitioning from zymogen to active proteases, is not solely dependent on DPP1. Thus, the killing of target cells by primary human CD8+ T cells, NK cells, and gene-engineered anti-CD19 CAR T cells was not blocked in vitro even after prior exposure to high concentrations of the reversible DPP1 inhibitor brensocatib. Consistent with this observation, the turnover of model granzyme A/B peptide substrates in the human CTL/NK cell lysates was not significantly reduced by brensocatib. In contrast, preincubation with brensocatib almost entirely abolished (>90%) both the cytotoxic activity of mouse CD8+ T cells and granzyme substrate turnover. Overall, our finding that the effects of DPP1 inhibition on human cytotoxic lymphocytes are attenuated in comparison to those of mice indicates that granzyme processing/activation pathways differ between mice and humans. Moreover, the in vitro data suggest that human subjects treated with reversible DPP1 inhibitors, such as brensocatib, are unlikely to experience any appreciable deficits in CTL/NK-cell-mediated immunities.

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. While a critical role for granzyme B in target cell apoptosis is undisputed, both it and the remaining granzymes also influence a variety of other biological processes (including important immunoregulatory functions), which are discussed in this review. This includes the targeting of many extracellular as well as intracellular substrates, and can also lead to deleterious outcomes for the host if granzyme expression or function are dysregulated or abrogated. A final important consideration is that granzyme repertoire, biochemistry and function vary considerably across species, probably resulting from the pressures applied by viruses and other pathogens across evolutionary time. This has implications for the interpretation of granzyme function in preclinical models of disease.

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. An immunosuppressant targeting this protein would provide the first-ever therapy focused specifically on one of the principal cell death pathways contributing to allotransplant rejection and underpinning multiple autoimmune and postinfectious diseases. No drugs that selectively block perforin-dependent cell death are currently in clinical use, so this perspective will review published novel small molecule inhibitors, concluding with in vivo proof-of-concept experiments performed in mouse models of perforin-mediated immune pathologies that provide a potential pathway toward a clinically useful therapeutic agent.

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

The mechanism of action of eprenetapopt (APR-246, PRIMA-1MET) 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 (SLC7A11, SHMT2, and MTHFD1L), as well as the enzymes required to synthesize glutathione (GCLC and GCLM), augments the activity of eprenetapopt. Eprenetapopt also inhibits iron-sulfur cluster biogenesis by limiting the cysteine desulfurase activity of NFS1, which potentiates ferroptosis and may restrict cellular proliferation. The combination of eprenetapopt with dietary serine and glycine restriction synergizes to inhibit esophageal xenograft tumor growth. These findings reframe the canonical view of eprenetapopt from a mutant-p53 reactivator to a ferroptosis inducer.

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 in vitro and to block the rejection of major histocompatibility complex (MHC)-mismatched mouse bone marrow cells. All compounds showed >99% binding to plasma proteins and demonstrated perforin inhibitory activity in vitro and in vivo. A lead compound, compound 1, that showed significant increases in allogeneic bone marrow preservation was evaluated for its plasma pharmacokinetics and in vivo efficacy at multiple dosing regimens to establish a pharmacokinetic/pharmacodynamic (PK/PD) relationship. The strongest PK/PD correlation was observed between perforin inhibition in vivo and time that total plasma concentrations remained above 900 µM, which correlates to unbound concentrations similar to 3× the unbound in vitro IC90 of compound 1. This PK/PD relationship will inform future dosing strategies of BZS perforin inhibitors to maintain concentrations above 3× the unbound IC90 for as long as possible to maximize efficacy and enhance progression toward clinical evaluation.

Severely impaired CTL killing is a feature of the neurological disorder Niemann-Pick disease type C1.

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-ß-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.

Differential cleavage of viral polypeptides by allotypic variants of granzyme B skews immunity to mouse cytomegalovirus.

We investigated the molecular basis for the remarkably different survival outcomes of mice expressing different alloforms of the pro-apoptotic serine protease granzyme B to mouse cytomegalovirus infection. Whereas C57BL/6 mice homozygous for granzyme BP (GzmBP/P) raise cytotoxic T lymphocytes that efficiently kill infected cells, those of C57BL/6 mice congenic for the outbred allele (GzmBW/W) fail to kill MCMV-infected cells and died from uncontrolled hepatocyte infection and acute liver failure. We identified subtle differences in how GzmBP and GzmBW activate cell death signalling - both alloforms predominantly activated pro-caspases directly, and cleaved pro-apoptotic Bid poorly. Consequently, neither alloform initiated mitochondrial outer membrane permeabilization, or was blocked by Bcl-2, Bcl-XL or co-expression of MCMV proteins M38.5/M41.1, which together stabilize mitochondria by sequestering Bak/Bax. Remarkably, mass spectrometric analysis of proteins from MCMV-infected primary mouse embryonic fibroblasts identified 13 cleavage sites in nine viral proteins (M18, M25, M28, M45, M80, M98, M102, M155, M164) that were cleaved >20-fold more efficiently by either GzmBP or GzmBW. Notably, M18, M28, M45, M80, M98, M102 and M164 were cleaved 20- >100-fold more efficiently by GzmBW, and so, would persist in infected cells targeted by CTLs from GzmBP/P mice. Conversely, M155 was cleaved >100-fold more efficiently by GzmBP, and would persist in cells targeted by CTLs of GzmBW/W mice. M25 was cleaved efficiently by both proteases, but at different sites. We conclude that different susceptibility to MCMV does not result from skewed endogenous cell death pathways, but rather, to as yet uncharacterised MCMV-intrinsic pathways that ultimately inhibit granzyme B-induced cell death.

ASC Modulates CTL Cytotoxicity and Transplant Outcome Independent of the Inflammasome.

The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1ß and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8+ T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11-deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8+ T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation.

Antigen-specific CD4+ CD25+ T cells induced by locally expressed ICOS-Ig: the role of Foxp3, Perforin, Granzyme B and IL-10 - an experimental study.

We have previously reported that ICOS-Ig expressed locally by a PIEC xenograft induces a perigraft cellular accumulation of CD4+ CD25+ Foxp3+ T cells and specific xenograft prolongation. In the present study we isolated and purified CD4+ CD25+ T cells from ICOS-Ig secreting PIEC grafts to examine their phenotype and mechanism of xenograft survival using knockout and mutant mice. CD4+ CD25+ T cells isolated from xenografts secreting ICOS-Ig were analysed by flow cytometry and gene expression by real-time PCR. Regulatory function was examined by suppression of xenogeneic or allogeneic primed CD4 T cells in vivo. Graft prolongation was shown to be dependent on a pre-existing Foxp3+ Treg, IL-10, perforin and granzyme B. CD4+ CD25+ Foxp3+ T cells isolated from xenografts secreting ICOS-Ig demonstrated a phenotype consistent with nTreg but with a higher expression of CD275 (ICOSL), expression of CD278 (ICOS) and MHC II and loss of CD73. Moreover, these cells were functional and specifically suppressed xenogeinic but not allogeneic primed T cells in vivo.

Serglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity.

The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means.