CD8⁺ T cells are key mediators of beta cell destruction in type 1 diabetes, employing the perforin/granzyme and Fas/Fas ligand pathways. Evidence from our studies and others indicates that perforin plays a dominant role in CD8⁺ T cell mediated beta cell destruction in non-obese diabetic (NOD) mice. As perforin is indispensable for delivering pro-apoptotic granzymes into the cytosol of target cells, pharmacological inhibition of perforin may potentially protect beta cells against CD8+ T cell cytotoxicity. Such a strategy will slow down beta cell loss and the progression to autoimmune diabetes. Small molecule perforin inhibitors recently becoming available provided an opportunity to test this hypothesis. The new small molecule perforin inhibitor SN34960 was evaluated for its ability to protect the beta cells of non-obese diabetic mice upon attack by autoreactive cytotoxic T lymphocytes, both in vitro and in vivo. In vitro, perforin inhibitor SN34960 protected isolated pancreatic islets from CD8+ T cell-mediated destruction (p<0.001). In vivo, a single dose of SN34960 reduced the death of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) peptide-loaded target cells by 50

The success of cancer immunotherapies is currently limited to a subset of patients, which underscores the urgent need to identify the processes by which tumours evade immunity. Through screening a kinome-wide CRISPR/Cas9 sgRNA library we identified MAP3K7 (TAK1) as a suppressor of CD8+ T cell mediated killing. We demonstrate that TAK1 acts as a cancer-intrinsic checkpoint by integrating signals from T cell-secreted TNF and IFNy effector cytokines to elicit a cytoprotective response. This cytoprotective response profoundly limits the anti-cancer activity these key effector molecules and completely abrogates bystander killing by perforin deficient T cells. Inhibition of the TAK1 checkpoint effectively redirects the combined TNF/IFNy pathway activation to promote inflammatory cell death via RIPK1 and Caspase-8 and simultaneously amplifies the output of the IFNy pathway, thereby priming cells for cytokine-induced cell death. Mechanistically, TAK1 deficiency led to proteasomal degradation of cFLIP, enhancing the formation of Complex II and subversion of other cytoprotective responses. Targeting the TAK1 checkpoint led to profound attenuation of tumour growth in immune competent mice, with minimal impact in immune deficient counterparts. Adoptive cell therapy led to preferential elimination of TAK1 deficient clones. Collectively, our study uncovers a cancer-intrinsic checkpoint controlled by TAK1 activity that switches TNF and IFNy responses from cytoprotective to apoptosis. Cancer cells exploit this to limit cell death in the presence of the cytotoxic lymphoctye cytokines TNF and IFNγ and therapeutic intervention can fully unleash the impact of these effector molecules both on the direct target and bystander cells. These findings highlight the clinical development of TAK1 biologics as a potential strategy to improve cancer immunotherapies through harnessing and enhancing the cytotoxic potential of CTL-derived cytokines.

Cancer immunotherapies benefit only a subset of patients, highlighting the need to define tumor-intrinsic mechanisms of immune evasion. Using a kinome-wide CRISPR-Cas9 screen, we identify MAP3K7 (transforming growth factor beta-activated kinase 1 [TAK1]) as a checkpoint that protects cancer cells from CD8+ T cell-mediated killing. TAK1 integrates tumor necrosis factor (TNF) and interferon gamma (IFNγ) signals to drive a cytoprotective response that blocks cytokine-induced death and prevents bystander killing by perforin-deficient T cells. Inhibition of TAK1 redirects TNF/IFNγ signaling toward apoptosis via RIPK1 and caspase-8 while simultaneously amplifying IFNγ outputs to further prime cells for cytokine-driven death. Mechanistically, TAK1 loss triggers proteasomal degradation of cFLIP, promoting complex II formation and undermining protective pathways. In immune-competent mice, TAK1 deficiency markedly impairs tumor growth, whereas immune-deficient hosts show little effect. Adoptive T cell therapy preferentially eliminates TAK1-deficient clones. These findings establish TAK1 as a tumor-intrinsic immune checkpoint and support TAK1 inhibition as a strategy to enhance cancer immunotherapy.

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.

Supplementary Figure S1: GVHD survival of lethally-irradiated (day 0, 1000cGy split dose) B6.WT, B6.ASC-/- or B6.NLRP3-/- recipients transplanted with BALB/c BM (5x106) and T cells (5x106). The non-GVHD control group received TCD BM cells (5x106) only. Results combined from 2 independent experiments (n = 20 per T cell-replete group, n = 8 for TCD BM group). ****P < 0.0001; B6.NLRP3-/- vs. B6.WT. Results are presented as means {plus minus} SEM.

Supplementary Table S1 (related to Fig. 4A): (A) Ingenuity Pathway Analysis derived from the uncorrected dataset displaying top canonical signaling pathways involving differentially regulated genes in recipients as described in Fig.4A. (B) 10 selected immunologically relevant genes identified as differentially regulated in mice transplanted with B6.WT or B6.ASC-/- grafts.

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.

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.

The mechanism of action of eprenetapopt (APR-246, PRIMA-1 MET ) 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.

The role of B-cell-activating factor (BAFF) in B-lymphocyte biology has been comprehensively studied, but its contributions to innate immunity remain unclear. Natural killer (NK) cells form the first line of defense against viruses and tumors, and have been shown to be defective in patients with systemic lupus erythematosus (SLE). The link between BAFF and NK cells in the development and progression of SLE remains unstudied. By assessing NK cell numbers in wild-type (WT), BAFF(-/-) (BAFF deficient), BAFF-R-/- (BAFF receptor deficient), TACI(-/-) (transmembrane activator and calcium modulator and cyclophilin ligand interactor deficient), BCMA(-/-) (B-cell maturation antigen deficient) and BAFF transgenic (Tg) mice, we observed that BAFF signaling through BAFF-R was essential for sustaining NK cell numbers in the spleen. However, according to the cell surface expression of CD27 and CD11b on NK cells, we found that BAFF was dispensable for NK cell maturation. Ex vivo and in vivo models showed that NK cells from BAFF(-/-) and BAFF Tg mice produced interferon-gamma and killed tumor cells at a level similar to that in WT mice. Finally, we established that NK cells do not express receptors that interact with BAFF in the steady state or in the BAFF Tg mouse model of SLE. Our findings demonstrate that BAFF has an indirect effect on NK cell homeostasis and no effect on NK cell function.

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.

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.

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.

AbstractThe 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 IL1β 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.

Background: We have previously reported that ICOS-Ig expressed locally by a graft 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 grafts to examine their phenotype, and used knockout and mutant mice to examine molecules involved in graft prolongation. Methods: The mechanism of xenograft survival observed with ICOS-Ig-secreting PIEC grafts was examined using selected knockout mice. CD4 + CD25 + T cells were isolated from xenografts secreting ICOS-Ig for phenotyping by flow cytometry, gene expression analysis by real-time PCR and regulatory function by suppression of xenogeneic or allogeneic primed CD4 T cells in vivo . Results: Graft prolongation is dependent on a pre-existing Foxp3 + Treg, IL-10, perforin and granzyme B. CD4 + CD25 + Foxp3 + T cells isolated from xenografts secreting ICOS-Ig have 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 are functional and specifically suppress primed T cells in vivo . Conclusions: Expression of soluble ICOS-Ig by a xenograft generates antigen specific CD4 + CD25 + Foxp3 + T cells that mediate specific graft prolongation using at least 3 key mediators: IL-10, perforin and granzyme B

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.

Whole-genome CRISPR screens identify resistance to TNF-mediated killing by T and NK cells as a tumor immune evasion mechanism.

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.