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Interleukin (IL)18 is a potent pro-inflammatory cytokine that is activated upon caspase 1 cleavage of the latent precursor, pro-IL18. Therapeutic T cell armoring with IL18 promotes autocrine stimulation and positive modulation of the tumor microenvironment (TME). However, existing strategies are imperfect since they involve constitutive/poorly regulated activity or fail to modify the TME. Here, we have substituted the caspase 1 cleavage site within pro-IL18 with that preferred by granzyme B, yielding GzB-IL18. We demonstrate that GzB-IL18 is constitutively released but remains functionally latent unless chimeric antigen receptor (CAR) T cells are activated, owing to concomitant granzyme B release. Armoring with GzB-IL18 enhances cytolytic activity, proliferation, interferon (IFN)-γ release, and anti-tumor efficacy by a similar magnitude to constitutively active IL18. We also demonstrate that GzB-IL18 provides a highly effective armoring strategy for γδ CAR T cells, leading to enhanced metabolic fitness and significant potentiation of therapeutic activity. Finally, we show that constitutively active IL18 can unmask CAR T cell-mediated cytokine release syndrome in immunocompetent mice. By contrast, GzB-IL18 promotes anti-tumor activity and myeloid cell re-programming without inducing such toxicity. Using this stringent system, we have tightly coupled the biological activity of IL18 to the activation state of the host CAR T cell, favoring safer clinical implementation of this technology., Competing Interests: Declaration of interests J.M. is founding scientist, shareholder, consultant, and Chief Scientific Officer of Leucid Bio. C.M.H., R.M., and D.M.D. are currently employees of Leucid Bio. J.M. and C.M.H. are named inventors on patent filings that pertain to granzyme B-regulated cytokines., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Extensive, large-scale single-cell profiling of healthy human blood at different ages is one of the critical pending tasks required to establish a framework for the systematic understanding of human aging. Here, using single-cell RNA/T cell receptor (TCR)/BCR-seq with protein feature barcoding, we profiled 317 samples from 166 healthy individuals aged 25-85 years old. From this, we generated a dataset from ∼2 million cells that described 55 subpopulations of blood immune cells. Twelve subpopulations changed with age, including the accumulation of GZMK + CD8 + T cells and HLA-DR + CD4 + T cells. In contrast to other T cell memory subsets, transcriptionally distinct NKG2C + GZMB - CD8 + T cells counterintuitively decreased with age. Furthermore, we found a concerted age-associated increase in type 2/interleukin (IL)4-expressing memory subpopulations across CD4 + and CD8 + T cell compartments (CCR4 + CD8 + Tcm and Th2 CD4 + Tmem), suggesting a systematic functional shift in immune homeostasis with age. Our work provides novel insights into healthy human aging and a comprehensive annotated resource., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genital herpes is characterized by recurrent episodes of epithelial blistering. The mechanisms causing this pathology are ill defined. Using a mouse model of vaginal herpes simplex virus 2 (HSV-2) infection, we show that interleukin-18 (IL-18) acts upon natural killer (NK) cells to promote accumulation of the serine protease granzyme B in the vagina, coinciding with vaginal epithelial ulceration. Genetic loss of granzyme B or therapeutic inhibition by a specific protease inhibitor reduces disease and restores epithelial integrity without altering viral control. Distinct effects of granzyme B and perforin deficiency on pathology indicates that granzyme B acts independent of its classic cytotoxic role. IL-18 and granzyme B are markedly elevated in human herpetic ulcers compared with non-herpetic ulcers, suggesting engagement of these pathways in HSV-infected patients. Our study reveals a role for granzyme B in destructing mucosal epithelium during HSV-2 infection, identifying a therapeutic target to augment treatment of genital herpes., Competing Interests: Declaration of interests D.J.G. is a co-founder and serves as the Chief Scientific Officer of viDA Therapeutics, which developed VTI-1002, the granzyme B inhibitor used in this study., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

CD8 T and NK cells mediate killing by delivery of perforin and granzyme B (GZB) stored in lysosome-like granules. We present a flow-cytometry-based protocol combined with a redirected killing assay to evaluate granule exocytosis and the cytotoxic potential of human CD8 T cells and NK cells. We describe the assessment of the delivered GZB inside the target cells. We then detail the detection of lysosome membrane protein CD107a exposed on the cell surface of the effector cells upon degranulation. For complete details on the use and execution of this protocol, please refer to Chen et al. (2021). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

γδ T cells are generally considered innate-like lymphocytes, however, an "adaptive-like" γδ compartment has now emerged. To understand transcriptional regulation of adaptive γδ T cell immunobiology, we combined single-cell transcriptomics, T cell receptor (TCR)-clonotype assignment, ATAC-seq, and immunophenotyping. We show that adult Vδ1 + T cells segregate into TCF7 + LEF1 + Granzyme B neg (T naive ) or T-bet + Eomes + BLIMP-1 + Granzyme B + (T effector ) transcriptional subtypes, with clonotypically expanded TCRs detected exclusively in T effector cells. Transcriptional reprogramming mirrors changes within CD8 + αβ T cells following antigen-specific maturation and involves chromatin remodeling, enhancing cytokine production and cytotoxicity. Consistent with this, in vitro TCR engagement induces comparable BLIMP-1, Eomes, and T-bet expression in naive Vδ1 + and CD8 + T cells. Finally, both human cytomegalovirus and Plasmodium falciparum infection in vivo drive adaptive Vδ1 T cell differentiation from T naive to T effector transcriptional status, alongside clonotypic expansion. Contrastingly, semi-invariant Vγ9 + Vδ2 + T cells exhibit a distinct "innate-effector" transcriptional program established by early childhood. In summary, adaptive-like γδ subsets undergo a pathogen-driven differentiation process analogous to conventional CD8 + T cells., Competing Interests: Declaration of interests M.J.T.S. has been employed by 10× Genomics since April 2018; this employment had no bearing on this work. The other authors declare no competing financial interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

αvβ8 integrin, a key activator of transforming growth factor β (TGF-β), inhibits anti-tumor immunity. We show that a potent blocking monoclonal antibody against αvβ8 (ADWA-11) causes growth suppression or complete regression in syngeneic models of squamous cell carcinoma, mammary cancer, colon cancer, and prostate cancer, especially when combined with other immunomodulators or radiotherapy. αvβ8 is expressed at the highest levels in CD4+CD25+ T cells in tumors, and specific deletion of β8 from T cells is as effective as ADWA-11 in suppressing tumor growth. ADWA-11 increases expression of a suite of genes in tumor-infiltrating CD8+ T cells normally inhibited by TGF-β and involved in tumor cell killing, including granzyme B and interferon-γ. The in vitro cytotoxic effect of tumor CD8 T cells is inhibited by CD4+CD25+ cells, and this suppressive effect is blocked by ADWA-11. These findings solidify αvβ8 integrin as a promising target for cancer immunotherapy., Competing Interests: Declaration of interests D.S. and R.J.A. were funded by a grant from the joint UCSF-Pfizer CTI. S.Y., A.G., K. Noorbehesht, B.Y., K. Niessen, and J.D.P. were employees of Pfizer. UCSF and Pfizer have filed a joint patent covering the use of a humanized version of ADWA-11 for cancer immunotherapy., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gasdermin B (GSDMB) belongs to a large family of pore-forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to human disease, its function in the immunological response to pathogens remains poorly understood. Here, we report a dynamic host-pathogen conflict between GSDMB and the IpaH7.8 effector protein secreted by enteroinvasive Shigella flexneri. We show that IpaH7.8 ubiquitinates and targets GSDMB for 26S proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of natural killer cells by suppressing granzyme-A-mediated activation of GSDMB. In contrast to the canonical function of most gasdermin family members, GSDMB does not inhibit Shigella by lysing host cells. Rather, it exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveal a mechanism employed by pathogens to counteract this host defense system., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Although T cell expansion depends on glycolysis, T effector cell differentiation requires signaling via the production of reactive oxygen species (ROS). Because the pentose phosphate pathway (PPP) regulates ROS by generating nicotinamide adenine dinucleotide phosphate (NADPH), we examined how PPP blockade affects T cell differentiation and function. Here, we show that genetic ablation or pharmacologic inhibition of the PPP enzyme 6-phosphogluconate dehydrogenase (6PGD) in the oxidative PPP results in the generation of superior CD8 + T effector cells. These cells have gene signatures and immunogenic markers of effector phenotype and show potent anti-tumor functions both in vitro and in vivo. In these cells, metabolic reprogramming occurs along with increased mitochondrial ROS and activated antioxidation machinery to balance ROS production against oxidative damage. Our findings reveal a role of 6PGD as a checkpoint for T cell effector differentiation/survival and evidence for 6PGD as an attractive metabolic target to improve tumor immunotherapy., Competing Interests: Declaration of interests V.A.B. has patents on the PD-1 pathway licensed by Bristol-Myers Squibb, Roche, Merck, EMD-Serono, Boehringer Ingelheim, AstraZeneca, Novartis, and Dako. The authors declare no other competing interests., (Published by Elsevier Inc.)

Systematic understanding of immune aging on a whole-body scale is currently lacking. We characterized age-associated alterations in immune cells across multiple mouse organs using single-cell RNA and antigen receptor sequencing and flow cytometry-based validation. We defined organ-specific and common immune alterations and identified a subpopulation of age-associated granzyme K (GZMK)-expressing CD8 + T (Taa) cells that are distinct from T effector memory (Tem) cells. Taa cells were highly clonal, had specific epigenetic and transcriptional signatures, developed in response to an aged host environment, and expressed markers of exhaustion and tissue homing. Activated Taa cells were the primary source of GZMK, which enhanced inflammatory functions of non-immune cells. In humans, proportions of the circulating GZMK + CD8 + T cell population that shares transcriptional and epigenetic signatures with mouse Taa cells increased during healthy aging. These results identify GZMK + Taa cells as a potential target to address age-associated dysfunctions of the immune system., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality., Competing Interests: Declaration of Interests The authors declare they have no competing interests., (Published by Elsevier Inc.)

If not properly regulated, the inflammatory immune response can promote carcinogenesis, as evident in colorectal cancer (CRC). Aiming to gain mechanistic insight into the link between inflammation and CRC, we perform transcriptomics analysis of human CRC, identifying a strong correlation between expression of the serine protease granzyme A (GzmA) and inflammation. In a dextran sodium sulfate and azoxymethane (DSS/AOM) mouse model, deficiency and pharmacological inhibition of extracellular GzmA both attenuate gut inflammation and prevent CRC development, including the initial steps of cell transformation and epithelial-to-mesenchymal transition. Mechanistically, extracellular GzmA induces NF-κB-dependent IL-6 production in macrophages, which in turn promotes STAT3 activation in cultured CRC cells. Accordingly, colon tissues from DSS/AOM-treated, GzmA-deficient animals present reduced levels of pSTAT3. By identifying GzmA as a proinflammatory protease that promotes CRC development, these findings provide information on mechanisms that link immune cell infiltration to cancer progression and present GzmA as a therapeutic target for CRC., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

T cell dysfunction is a characteristic feature of chronic viral infection and cancer. Recent studies in chronic lymphocytic choriomeningitis virus (LCMV) infection have defined a PD-1 + Tcf-1 + CD8 + T cell subset capable of self-renewal and differentiation into more terminally differentiated cells that downregulate Tcf-1 and express additional inhibitory molecules such as Tim3. Here, we demonstrated that expression of the glycoprotein CD101 divides this terminally differentiated population into two subsets. Stem-like Tcf-1 + CD8 + T cells initially differentiated into a transitory population of CD101 - Tim3 + cells that later converted into CD101 + Tim3 + cells. Recently generated CD101 - Tim3 + cells proliferated in vivo, contributed to viral control, and were marked by an effector-like transcriptional signature including expression of the chemokine receptor CX3CR1, pro-inflammatory cytokines, and granzyme B. PD-1 pathway blockade increased the numbers of CD101 - Tim3 + CD8 + T cells, suggesting that these newly generated transitional cells play a critical role in PD-1-based immunotherapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

T cell recognition of specific antigens mediates protection from pathogens and controls neoplasias, but can also cause autoimmunity. Our knowledge of T cell antigens and their implications for human health is limited by the technical limitations of T cell profiling technologies. Here, we present T-Scan, a high-throughput platform for identification of antigens productively recognized by T cells. T-Scan uses lentiviral delivery of antigen libraries into cells for endogenous processing and presentation on major histocompatibility complex (MHC) molecules. Target cells functionally recognized by T cells are isolated using a reporter for granzyme B activity, and the antigens mediating recognition are identified by next-generation sequencing. We show T-Scan correctly identifies cognate antigens of T cell receptors (TCRs) from viral and human genome-wide libraries. We apply T-Scan to discover new viral antigens, perform high-resolution mapping of TCR specificity, and characterize the reactivity of a tumor-derived TCR. T-Scan is a powerful approach for studying T cell responses., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Malaria represents a major cause of death from infectious disease. Hemozoin is a Plasmodium-derived product that contributes to progression of cerebral malaria. However, there is a gap of knowledge regarding how hemozoin is recognized by innate immunity. Myeloid C-type lectin receptors (CLRs) encompass a family of carbohydrate-binding receptors that act as pattern recognition receptors in innate immunity. In the present study, we identify the CLR CLEC12A as a receptor for hemozoin. Dendritic cell-T cell co-culture assays indicate that the CLEC12A/hemozoin interaction enhances CD8 + T cell cross-priming. Using the Plasmodium berghei Antwerpen-Kasapa (ANKA) mouse model of experimental cerebral malaria (ECM), we find that CLEC12A deficiency protects mice from ECM, illustrated by reduced ECM incidence and ameliorated clinical symptoms. In conclusion, we identify CLEC12A as an innate sensor of plasmodial hemozoin., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Adoptive cell therapy with chimeric antigen receptor (CAR)-redirected T cells induced spectacular regressions of leukemia and lymphoma, however, failed so far in the treatment of solid tumors. A cause is thought to be T cell repression through TGF-β, which is massively accumulating in the tumor tissue. Here, we show that T cells with a CD28-ζ CAR, but not with a 4-1BB-ζ CAR, resist TGF-β-mediated repression. Mechanistically, LCK activation and consequently IL-2 release and autocrine IL-2 receptor signaling mediated TGF-β resistance; deleting the LCK-binding motif in the CD28 CAR abolished both IL-2 secretion and TGF-β resistance, while IL-2 add-back restored TGF-β resistance. Other γ-cytokines like IL-7 and IL-15 could replace IL-2 in this context. This is demonstrated by engineering IL-2 deficient CD28ΔLCK-ζ CAR T cells with a hybrid IL-7 receptor to provide IL-2R β chain signaling upon IL-7 binding. Such modified T cells showed improved CAR T cell activity against TGF-β + tumors. Data draw the concept that an autocrine loop resulting in IL-2R signaling can make CAR T cells more potent in staying active against TGF-β + solid tumors., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Human cytotoxic lymphocytes kill intracellular microbes. The cytotoxic granule granzyme proteases released by cytotoxic lymphocytes trigger oxidative bacterial death by disrupting electron transport, generating superoxide anion and inactivating bacterial oxidative defenses. However, they also cause non-oxidative cell death because anaerobic bacteria are also killed. Here, we use differential proteomics to identify granzyme B substrates in three unrelated bacteria: Escherichia coli, Listeria monocytogenes, and Mycobacteria tuberculosis. Granzyme B cleaves a highly conserved set of proteins in all three bacteria, which function in vital biosynthetic and metabolic pathways that are critical for bacterial survival under diverse environmental conditions. Key proteins required for protein synthesis, folding, and degradation are also substrates, including multiple aminoacyl tRNA synthetases, ribosomal proteins, protein chaperones, and the Clp system. Because killer cells use a multipronged strategy to target vital pathways, bacteria may not easily become resistant to killer cell attack., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We recently found that a unique subset of innate-like γδ T cells develops from the DN2a stage of the fetal thymus independently of the zinc-finger transcription factor B cell leukemia/lymphoma 11b (Bcl11b). Herein, we characterize these Bcl11b-independent γδ T cells in the periphery as CD5 - NK1.1 + and Granzyme B + , and we show that they are capable of producing interferon (IFN)-γ upon T cell receptor stimulation without Ca 2+ influx. In wild-type mice, these cells were sparse in lymphoid tissues but abundant in non-lymphoid tissues, such as the liver. Bcl11b-independent CD5 - NK1.1 + γδ T cells appeared and contributed to early protection before Bcl11b-dependent CD5 + NK1.1 - γδ T cells following Listeria monocytogenes infection, resembling their sequential appearance during development in the thymus., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Cytotoxic T lymphocytes (CTLs) used in immunotherapy are typically cultured under atmospheric O 2 pressure but encounter hypoxic conditions inside tumors. Activating CTLs under hypoxic conditions has been shown to improve their cytotoxicity in vitro, but the mechanism employed and the implications for immunotherapy remain unknown. We activated and cultured OT-I CD8 T cells at either 1% or 20% O 2 . Hypoxic CTLs survived, as well as normoxic ones, in vitro but killed OVA-expressing B16 melanoma cells more efficiently. Hypoxic CTLs contained similar numbers of cytolytic granules and released them as efficiently but packaged more granzyme-B in each granule without producing more perforin. We imaged CTL distribution and motility inside B16-OVA tumors using confocal and intravital 2-photon microscopy and observed no obvious differences. However, mice treated with hypoxic CTLs exhibited better tumor regression and survived longer. Thus, hypoxic CTLs may perform better in tumor immunotherapy because of higher intrinsic cytotoxicity rather than improved migration inside tumors., (Copyright © 2017 Elsevier Inc. All rights reserved.)

For more than 20 years perforin and granzymes (GZMs) have been recognized as key cell death executors of cytotoxic T (Tc) and natural killer (NK) cells during cancer immunosurveillance. In immune surveillance, perforin and GZMB, the most potent cytotoxic molecules, act mainly as antitumoral and anti-infectious factors. However, when expressed by immune regulatory cells they may contribute to immune evasion of specific cancer types. By contrast, the other major granzyme, GZMA, seems not to play a major role in Tc/NK cell-mediated cytotoxicity, but acts as a proinflammatory cytokine that might contribute to cancer development. Members of the GZM family also regulate other biological processes unrelated to cell death, such as angiogenesis, vascular integrity, extracellular matrix remodeling, and barrier function, all of which contribute to cancer initiation and progression. Thus, a new paradigm is emerging in the field of oncoimmunology. Can GZMs act as protumoral factors under some circumstances? We review the diverse roles of GZMs in cancer progression, and new therapeutic opportunities emerging from targeting these protumoral roles., (Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.)

CD4 + T cells develop distinct and often contrasting helper, regulatory, or cytotoxic activities. Typically a property of CD8 + T cells, granzyme-mediated cytotoxic T cell (CTL) potential is also exerted by CD4 + T cells. However, the conditions that induce CD4 + CTLs are not entirely understood. Using single-cell transcriptional profiling, we uncover a unique signature of Granzyme B (GzmB) + CD4 + CTLs, which distinguishes them from other CD4 + T helper (Th) cells, including Th1 cells, and strongly contrasts with the follicular helper T (Tfh) cell signature. The balance between CD4 + CTL and Tfh differentiation heavily depends on the class of infecting virus and is jointly regulated by the Tfh-related transcription factors Bcl6 and Tcf7 (encoding TCF-1) and by the expression of the inhibitory receptors PD-1 and LAG3. This unique profile of CD4 + CTLs offers targets for their study, and its antagonism by the Tfh program separates CD4 + T cells with either helper or killer functions., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Malignancy can be suppressed by the immune system in a process termed immunosurveillance. However, to what extent immunosurveillance occurs in spontaneous cancers and the composition of participating cell types remains obscure. Here, we show that cell transformation triggers a tissue-resident lymphocyte response in oncogene-induced murine cancer models. Non-circulating cytotoxic lymphocytes, derived from innate, T cell receptor (TCR)αβ, and TCRγδ lineages, expand in early tumors. Characterized by high expression of NK1.1, CD49a, and CD103, these cells share a gene-expression signature distinct from those of conventional NK cells, T cells, and invariant NKT cells. Generation of these lymphocytes is dependent on the cytokine IL-15, but not the transcription factor Nfil3 that is required for the differentiation of tumor-infiltrating NK cells, and IL-15 deficiency, but not Nfil3 deficiency, results in accelerated tumor growth. These findings reveal a tumor-elicited immunosurveillance mechanism that engages unconventional type-1-like innate lymphoid cells and type 1 innate-like T cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Immune memory cells are poised to rapidly expand and elaborate effector functions upon reinfection yet exist in a functionally quiescent state. The paradigm is that memory T cells remain inactive due to lack of T cell receptor (TCR) stimuli. Here, we report that regulatory T (Treg) cells orchestrate memory T cell quiescence by suppressing effector and proliferation programs through inhibitory receptor, cytotoxic-T-lymphocyte-associated protein-4 (CTLA-4). Loss of Treg cells resulted in activation of genome-wide transcriptional programs characteristic of effector T cells and drove transitioning as well as established memory CD8(+) T cells toward terminally differentiated KLRG-1(hi)IL-7Rα(lo)GzmB(hi) phenotype, with compromised metabolic fitness, longevity, polyfunctionality, and protective efficacy. CTLA-4 functionally replaced Treg cells in trans to rescue memory T cell defects and restore homeostasis. These studies present the CTLA-4-CD28-CD80/CD86 axis as a potential target to accelerate vaccine-induced immunity and improve T cell memory quality in current cancer immunotherapies proposing transient Treg cell ablation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Granzyme B released by leukocytes cleaves multiple intracellular substrates required for target cell lysis. In this issue of Immunity, Prakash et al. (2014) demonstrate that granzyme B cleaves basement membrane proteins and promotes cytotoxic T cell diapedesis into inflamed tissue., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Granzyme B (GzmB) is a protease with a well-characterized intracellular role in targeted destruction of compromised cells by cytotoxic lymphocytes. However, GzmB also cleaves extracellular matrix components, suggesting that it influences the interplay between cytotoxic lymphocytes and their environment. Here, we show that GzmB-null effector T cells and natural killer (NK) cells exhibited a cell-autonomous homing deficit in mouse models of inflammation and Ectromelia virus infection. Intravital imaging of effector T cells in inflamed cremaster muscle venules revealed that GzmB-null cells adhered normally to the vessel wall and could extend lamellipodia through it but did not cross it efficiently. In vitro migration assays showed that active GzmB was released from migrating cytotoxic lymphocytes and enabled chemokine-driven movement through basement membranes. Finally, proteomic analysis demonstrated that GzmB cleaved basement membrane constituents. Our results highlight an important role for GzmB in expediting cytotoxic lymphocyte diapedesis via basement membrane remodeling., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Bacterial components are recognized by the immune system through activation of the inflammasome, eventually causing processing of the proinflammatory cytokine interleukin-1? (IL-1?), a pleiotropic cytokine and one of the most important mediators of inflammation, through the protease caspase-1. Synthesis of the precursor protein and processing into its bioactive form are tightly regulated, given that disturbed control of IL-1? release can cause severe autoinflammatory diseases or contribute to cancer development. We show that the bacterial Pasteurella multocida toxin (PMT) triggers Il1b gene transcription in macrophages independently of Toll-like receptor signaling through RhoA/Rho-kinase-mediated NF-?? activation. Furthermore, PMT mediates signal transducer and activator of transcription (STAT) protein-controlled granzyme A (a serine protease) expression in macrophages. The exocytosed granzyme A enters target cells and mediates IL-1? maturation independently of caspase-1 and without inducing cytotoxicity. These findings show that macrophages can induce an IL-1?-initiated immune response independently of inflammasome activity.

Immune control of infections with viruses or intracellular bacteria relies on cytotoxic CD8(+) T cells that use granzyme B (GzmB) for elimination of infected cells. During inflammation, mature antigen-presenting dendritic cells instruct naive T cells within lymphoid organs to develop into effector T cells. Here, we report a mechanistically distinct and more rapid process of effector T cell development occurring within 18 hr. Such rapid acquisition of effector T cell function occurred through cross-presenting liver sinusoidal endothelial cells (LSECs) in the absence of innate immune stimulation and known costimulatory signaling. Rather, interleukin-6 (IL-6) trans-signaling was required and sufficient for rapid induction of GzmB expression in CD8(+) T cells. Such LSEC-stimulated GzmB-expressing CD8(+) T cells further responded to inflammatory cytokines, eliciting increased and protracted effector functions. Our findings identify a role for IL-6 trans-signaling in rapid generation of effector function in CD8(+) T cells that may be beneficial for vaccination strategies., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

During bacterial sepsis, proinflammatory cytokines contribute to multiorgan failure and death in a process regulated in part by cytolytic cell granzymes. When challenged with a sublethal dose of the identified mouse pathogen Brucella microti, wild-type (WT) and granzyme A (gzmA)(-/-) mice eliminate the organism from liver and spleen in 2 or 3 weeks, whereas the bacteria persist in mice lacking perforin or granzyme B as well as in mice depleted of Tc cells. In comparison, after a fatal challenge, only gzmA(-/-) mice exhibit increased survival, which correlated with reduced proinflammatory cytokines. Depletion of natural killer (NK) cells protects WT mice from sepsis without influencing bacterial clearance and the transfer of WT, but not gzmA(-/-) NK, cells into gzmA(-/-) recipients restores the susceptibility to sepsis. Therefore, infection-related pathology, but not bacterial clearance, appears to require gzmA, suggesting the protease may be a therapeutic target for the prevention of bacterial sepsis without affecting immune control of the pathogen., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

When killer lymphocytes recognize infected cells, perforin delivers cytotoxic proteases (granzymes) into the target cell to trigger apoptosis. What happens to intracellular bacteria during this process is unclear. Human, but not rodent, cytotoxic granules also contain granulysin, an antimicrobial peptide. Here, we show that granulysin delivers granzymes into bacteria to kill diverse bacterial strains. In Escherichia coli, granzymes cleave electron transport chain complex I and oxidative stress defense proteins, generating reactive oxygen species (ROS) that rapidly kill bacteria. ROS scavengers and bacterial antioxidant protein overexpression inhibit bacterial death. Bacteria overexpressing a GzmB-uncleavable mutant of the complex I subunit nuoF or strains that lack complex I still die, but more slowly, suggesting that granzymes disrupt multiple vital bacterial pathways. Mice expressing transgenic granulysin are better able to clear Listeria monocytogenes. Thus killer cells play an unexpected role in bacterial defense., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Immunological control of infections or tumors depends on the release of effector cytokines and polarized secretion of cytotoxic granules from T cells and natural killer cells. Here we show that the sorting receptor Sortilin controlled both processes. In murine Sortilin-deficient cytotoxic T lymphocytes, regulated secretion of granzyme A and cytotoxic killing was enhanced and correlated with increased vesicle-associated membrane protein 7 availability. In contrast, loss of Sortilin reduced the release of interferon-γ upon infections and in autoimmune colitis. Exit of interferon-γ from the Golgi apparatus required the presence of Sortilin. Furthermore, we tracked the transport route of interferon-γ beyond this Sortilin-dependent Golgi to early endosome step. In wild-type T cells, trafficking of interferon-γ from the endosomal sorting platform to the plasma membrane proceeded independently of recycling endosomes, and interferon-γ remained excluded from late endosomes. Our results suggest that Sortilin modulates systemic immune responses through exocytic sorting of immunological effector molecules., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Tumor immune surveillance and cancer immunotherapies are thought to depend on the intratumoral infiltration of activated CD8(+) T cells. Intratumoral CD8(+) T cells are rare and lack activity. IL-10 is thought to contribute to the underlying immune suppressive microenvironment. Defying those expectations we demonstrate that IL-10 induces several essential mechanisms for effective antitumor immune surveillance: infiltration and activation of intratumoral tumor-specific cytotoxic CD8(+) T cells, expression of the Th1 cytokine interferon-γ (IFNγ) and granzymes in CD8(+) T cells, and intratumoral antigen presentation molecules. Consequently, tumor immune surveillance is weakened in mice deficient for IL-10 whereas transgenic overexpression of IL-10 protects mice from carcinogenesis. Treatment with pegylated IL-10 restores tumor-specific intratumoral CD8(+) T cell function and controls tumor growth., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Granzyme B is a cytotoxic lymphocyte-derived protease that plays a central role in promoting apoptosis of virus-infected target cells, through direct proteolysis and activation of constituents of the cell death machinery. However, previous studies have also implicated granzymes A and B in the production of proinflammatory cytokines, via a mechanism that remains undefined. Here we show that IL-1α is a substrate for granzyme B and that proteolysis potently enhanced the biological activity of this cytokine in vitro as well as in vivo. Consistent with this, compared with full-length IL-1α, granzyme B-processed IL-1α exhibited more potent activity as an immunoadjuvant in vivo. Furthermore, proteolysis of IL-1α within the same region, by proteases such as calpain and elastase, was also found to enhance its biological potency. Thus, IL-1α processing by multiple immune-related proteases, including granzyme B, acts as a switch to enhance the proinflammatory properties of this cytokine., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Type III/lambda interferons (IFNs) were discovered less than a decade ago and are still in the process of being characterized. Although previous studies have focused on the function of IFN-lambda 3 (also known as interleukin (IL)-28B) in a small animal model, it is unknown whether these functions would translate to a larger, more relevant model. Thus in the present study, we have used DNA vaccination as a method of studying the influence of IFN-lambda 3 on adaptive immune responses in rhesus macaques. Results of our study show for the first time that IFN-lambda 3 has significant influence on antigen-specific CD8(+) T-cell function, especially in regards to cytotoxicity. Peripheral CD8(+) T cells from animals that were administered IFN-lambda 3 showed substantially increased cytotoxic responses as gauged by CD107a and granzyme B coexpression as well as perforin release. Moreover, CD8(+) T cells isolated from the mesenteric lymph nodes (MLN) of animals receiving IFN-lambda 3 loaded significant amounts of granzyme B upon extended antigenic stimulation and induced significantly more granzyme B-mediated cell death of peptide pulsed targets. These data suggest that IFN-lambda 3 is a potent effector of the immune system with special emphasis on CD8(+) T-cell killing functions which warrants further study as a possible immunoadjuvant.

The characteristics, importance, and molecular requirements for interactions between mast cells (MCs) and CD8(+) T cells have not been elucidated. Here, we demonstrated that MCs induced antigen-specific CD8(+) T cell activation and proliferation. This process required direct cell contact and MHC class I-dependent antigen cross-presentation by MCs and induced the secretion of interleukin-2, interferon-gamma, and macrophage inflammatory protein-1alpha by CD8(+) T cells. MCs regulated antigen-specific CD8(+) T cell cytotoxicity by increasing granzyme B expression and by promoting CD8(+) T cell degranulation. Because MCs also upregulated their expression of costimulatory molecules (4-1BB) and released osteopontin upon direct T cell contact, MC-T cell interactions probably are bidirectional. In vivo, adoptive transfer of antigen-pulsed MCs induced MHC class I-dependent, antigen-specific CD8(+) T cell proliferation, and MCs regulated CD8(+) T cell-specific priming in experimental autoimmune encephalomyelitis. Thus, MCs are important players in antigen-specific regulation of CD8(+) T cells.

T cell exhaustion is common during chronic infections and can prevent optimal immunity. Although recent studies have demonstrated the importance of inhibitory receptors and other pathways in T cell exhaustion, the underlying transcriptional mechanisms are unknown. Here, we define a role for the transcription factor Blimp-1 in CD8(+) T cell exhaustion during chronic viral infection. Blimp-1 repressed key aspects of normal memory CD8(+) T cell differentiation and promoted high expression of inhibitory receptors during chronic infection. These cardinal features of CD8(+) T cell exhaustion were corrected by conditionally deleting Blimp-1. Although high expression of Blimp-1 fostered aspects of CD8(+) T cell exhaustion, haploinsufficiency indicated that moderate Blimp-1 expression sustained some effector function during chronic viral infection. Thus, we identify Blimp-1 as a transcriptional regulator of CD8(+) T cell exhaustion during chronic viral infection and propose that Blimp-1 acts as a transcriptional rheostat balancing effector function and T cell exhaustion.

Adoptive cytotoxic T lymphocyte (CTL) therapy has an important implication in treating cancer patients. Here, we investigate whether adoptive transfer of human papillomavirus (HPV) E7-specific CTL can enhance tumor chemoresponse using an established cervical cancer animal model. Cisplatin-based chemotherapy plus CTL therapy showed an improved therapeutic effectiveness, along with antitumor protective responses to a parental tumor cell rechallenge. Cisplatin treatment dose-dependently increased the expression of Fas, intercellular adhesion molecule (ICAM)-1, and major histocompatibility complex (MHC) class I antigens (Ags) on tumor cells in vitro. However, CTL-expressing FasL failed to improve antitumor activity in vitro and in animals, resulting from nonfunctional Fas expressed on tumor cells. In contrast, ethylene glycol tetraacetic acid (EGTA) treatment blocked increased sensitivity of cisplatin-treated tumor cells to CTL-mediated killing in vitro, suggesting an important role of the perforin/granzyme-mediated pathway for improved therapeutic effectiveness. This notion was further confirmed by perforin knockout animal studies. Thus, this study shows that (i) modulation of Ag (Fas, ICAM-1) expression by tumor cells has little effect on their increased sensitivity to CTL-mediated killing, (ii) improved therapeutic effectiveness is mediated mainly through the perforin/granzyme-mediated tumor killing pathway, and (iii) a combination of chemotherapy and adoptive E7-specific CTL transfer augments antitumor therapeutic activity in vivo. This finding may have important implications for treating HPV-associated cervical cancer.

Perforin, a pore-forming protein secreted by cytotoxic lymphocytes, is indispensable for destroying virus-infected cells and for maintaining immune homeostasis. Perforin polymerizes into transmembrane channels that inflict osmotic stress and facilitate target cell uptake of proapoptotic granzymes. Despite this, the mechanism through which perforin monomers self-associate remains unknown. Our current study establishes the molecular basis for perforin oligomerization and pore assembly. We show that after calcium-dependent membrane binding, direct ionic attraction between the opposite faces of adjacent perforin monomers was necessary for pore formation. By using mutagenesis, we identified the opposing charges on residues Arg213 (positive) and Glu343 (negative) to be critical for intermolecular interaction. Specifically, disrupting this interaction had no effect on perforin synthesis, folding, or trafficking in the killer cell, but caused a marked kinetic defect of oligomerization at the target cell membrane, severely disrupting lysis and granzyme B-induced apoptosis. Our study provides important insights into perforin's mechanism of action.

Virus-specific CD8+ T cells probably mediate control over HIV replication in rare individuals, termed long-term nonprogressors (LTNPs) or elite controllers. Despite extensive investigation, the mechanisms responsible for this control remain incompletely understood. We observed that HIV-specific CD8+ T cells of LTNPs persisted at higher frequencies than those of treated progressors with equally low amounts of HIV. Measured on a per-cell basis, HIV-specific CD8+ T cells of LTNPs efficiently eliminated primary autologous HIV-infected CD4+ T cells. This function required lytic granule loading of effectors and delivery of granzyme B to target cells. Defective cytotoxicity of progressor effectors could be restored after treatment with phorbol ester and calcium ionophore. These results establish an effector function and mechanism that clearly segregate with immunologic control of HIV. They also demonstrate that lytic granule contents of memory cells are a critical determinant of cytotoxicity that must be induced for maximal per-cell killing capacity.

Rare HIV-1-infected individuals are able to control viral replication without antiretroviral therapy. In this issue of Immunity, Migueles et al. (2008) show that HIV-1-specific CD8+ T cells from these individuals effectively suppress viral replication by granzyme B-mediated killing of target cells.

Myc activity is emerging as a key element in acquisition and maintenance of stem cell properties. We have previously shown that c-Myc deficiency results in accumulation of defective hematopoietic stem cells (HSCs) due to niche-dependent differentiation defects. Here we report that immature HSCs coexpress c-myc and N-myc mRNA at similar levels. Although conditional deletion of N-myc in the bone marrow does not affect hematopoiesis, combined deficiency of c-Myc and N-Myc (dKO) results in pancytopenia and rapid lethality. Interestingly, proliferation of HSCs depends on both myc genes during homeostasis, but is c-Myc/N-Myc independent during bone marrow repair after injury. Strikingly, while most dKO hematopoietic cells undergo apoptosis, only self-renewing HSCs accumulate the cytotoxic molecule Granzyme B, normally employed by the innate immune system, thereby revealing an unexpected mechanism of stem cell apoptosis. Collectively, Myc activity (c-Myc and N-Myc) controls crucial aspects of HSC function including proliferation, differentiation, and survival.

c-myc is a key regulator of hemopoietic stem cell (HSC) activity. In this issue of Cell Stem Cell, Laurenti et al. (2008) show that c-myc and N-myc control HSC survival and link this finding to the regulation of granzyme B expression.

In this issue of Immunity, Metkar et al. (2008) present evidence that granzyme A plays a role in inflammatory signaling and that contrary to previous studies, it is incapable of inducing target cell death. The work challenges us to reconsider the broader biological roles of all the granzymes.

Granzyme A (GzmA) is considered a major proapoptotic protease. We have discovered that GzmA-induced cell death involves rapid membrane damage that depends on the synergy between micromolar concentrations of GzmA and sublytic perforin (PFN). Ironically, GzmA and GzmB, independent of their catalytic activity, both mediated this swift necrosis. Even without PFN, lower concentrations of human GzmA stimulated monocytic cells to secrete proinflammatory cytokines (interleukin-1beta [IL-1beta], TNFalpha, and IL-6) that were blocked by a caspase-1 inhibitor. Moreover, murine GzmA and GzmA(+) cytotoxic T lymphocytes (CTLs) induce IL-1beta from primary mouse macrophages, and GzmA(-/-) mice resist lipopolysaccharide-induced toxicity. Thus, the granule secretory pathway plays an unexpected role in inflammation, with GzmA acting as an endogenous modulator.

Little is known about the functional differences between the human skin myeloid dendritic cell (DC) subsets, epidermal CD207(+) Langerhans cells (LCs) and dermal CD14(+) DCs. We showed that CD14(+) DCs primed CD4(+) T cells into cells that induce naive B cells to switch isotype and become plasma cells. In contrast, LCs preferentially induced the differentiation of CD4(+) T cells secreting T helper 2 (Th2) cell cytokines and were efficient at priming and crosspriming naive CD8(+) T cells. A third DC population, CD14(-)CD207(-)CD1a(+) DC, which resides in the dermis, could activate CD8(+) T cells better than CD14(+) DCs but less efficiently than LCs. Thus, the human skin displays three DC subsets, two of which, i.e., CD14(+) DCs and LCs, display functional specializations, the preferential activation of humoral and cellular immunity, respectively.

The induction of caspase-independent cell death by killer lymphocytes involves the serine protease granzyme A (GzmA). In this issue, Martinvalet et al. (2008) show that GzmA penetrates the mitochondrial matrix without perturbing normal mitochondrial functions. In the mitochondrial matrix, GzmA cleaves NDUFS3 (a component of the electron transport chain) leading to production of reactive oxygen species and ultimately to cell death.

The killer lymphocyte protease granzyme A (GzmA) triggers caspase-independent target cell death with morphological features of apoptosis. We previously showed that GzmA acts directly on mitochondria to generate reactive oxygen species (ROS) and disrupt the transmembrane potential (DeltaPsi(m)) but does not permeabilize the mitochondrial outer membrane. Mitochondrial damage is critical to GzmA-induced cell death since cells treated with superoxide scavengers are resistant to GzmA. Here we find that GzmA accesses the mitochondrial matrix to cleave the complex I protein NDUFS3, an iron-sulfur subunit of the NADH:ubiquinone oxidoreductase complex I, after Lys56 to interfere with NADH oxidation and generate superoxide anions. Target cells expressing a cleavage site mutant of NDUFS3 are resistant to GzmA-mediated cell death but remain sensitive to GzmB.

Ischemia and seizure cause excessive neuronal excitation that is associated with brain acidosis and neuronal cell death. However, the molecular mechanism of acidification-triggered neuronal injury is incompletely understood. Here, we show that asparagine endopeptidase (AEP) is activated under acidic condition, cuts SET, an inhibitor of DNase, and triggers DNA damage in brain, which is inhibited by PIKE-L. SET, a substrate of caspases, was cleaved by acidic cytosolic extract independent of caspase activation. Fractionation of the acidic cellular extract yielded AEP that is required for SET cleavage. We found that kainate provoked AEP activation and SET cleavage at N175, triggering DNA nicking in wild-type, but not AEP null, mice. PIKE-L strongly bound SET and prevented its degradation by AEP, leading to resistance of neuronal cell death. Moreover, AEP also mediated stroke-provoked SET cleavage and cell death in brain. Thus, AEP might be one of the proteinases activated by acidosis triggering neuronal injury during neuroexcitotoxicity or ischemia.

Granzyme B is important for the ability of NK cells and CD8(+) T cells to kill their targets. However, we showed here that granzyme B-deficient mice clear both allogeneic and syngeneic tumor cell lines more efficiently than do wild-type (WT) mice. To determine whether regulatory T (Treg) cells utilize granzyme B to suppress immune responses against these tumors, we examined the expression and function of granzyme B in Treg cells. Granzyme B was not expressed in naive Treg cells but was highly expressed in 5%-30% of CD4(+)Foxp3(+) Treg cells in the tumor environment. Adoptive transfer of WT Treg cells, but not granzyme B- or perforin-deficient Treg cells, into granzyme B-deficient mice partially restored susceptibility to tumor growth; Treg cells derived from the tumor environment could induce NK and CD8(+) T cell death in a granzyme B- and perforin-dependent fashion. Granzyme B and perforin are therefore relevant for Treg cell-mediated suppression of tumor clearance in vivo.

Although activated murine NK cells can use the granule exocytosis pathway to kill target cells immediately upon recognition, resting murine NK cells are minimally cytotoxic for unknown reasons. Here, we showed that resting NK cells contained abundant granzyme A, but little granzyme B or perforin; in contrast, the mRNAs for all three genes were abundant. Cytokine-induced in vitro activation of NK cells resulted in potent cytotoxicity associated with a dramatic increase in granzyme B and perforin, but only minimal changes in mRNA abundance for these genes. The same pattern of regulation was found in vivo with murine cytomegalovirus infection as a physiologic model of NK cell activation. These data suggest that resting murine NK cells are minimally cytotoxic because of a block in perforin and granzyme B mRNA translation that is released by NK cell activation.