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Loss of Bid confers clonogenic survival to granzyme B-treated cells, however the exact role of Bid-induced mitochondrial damage – upstream or downstream of caspases – remains controversial. Here we show that direct cleavage of Bid by granzyme B, but not caspases, was required for granzyme B-induced apoptosis. Release of cytochrome c and SMAC, but not AIF or endonuclease G, occurred in the absence of caspase activity and correlated with the onset of apoptosis and loss of clonogenic potential. Loss of mitochondrial trans-membrane potential (ΔΨm) was also caspase independent, however if caspase activity was blocked the mitochondria regenerated their ΔΨm. Loss of ΔΨm was not required for rapid granzyme B-induced apoptosis and regeneration of ΔΨm following cytochrome c release did not confer clonogenic survival. This functional dissociation of cytochrome c and SMAC release from loss of ΔΨm demonstrates the essential contribution of Bid upstream of caspase activation during granzyme B-induced apoptosis.Cell Death and Differentiation (2006) 13, 607–618. doi:10.1038/sj.cdd.4401772; published online 16 September 2005 [ABSTRACT FROM AUTHOR]

Various microphone array geometries (e.g., linear, circular, square, cubic, spherical, etc.) have been used to improve the positioning accuracy of sound source localization. However, whether these array structures are optimal for various specific localization scenarios is still a subject of debate. This paper addresses a microphone array optimization method for sound source localization based on TDOA (time difference of arrival). The geometric structure of the microphone array is established in parametric form. A triangulation method with TDOA was used to build the spatial sound source location model, which consists of a group of nonlinear multivariate equations. Through reasonable transformation, the nonlinear multivariate equations can be converted to a group of linear equations that can be approximately solved by the weighted least square method. Then, an optimization model based on particle swarm optimization (PSO) algorithm was constructed to optimize the geometric parameters of the microphone array under different localization scenarios combined with the spatial sound source localization model. In the optimization model, a reasonable fitness evaluation function is established which can comprehensively consider the positioning accuracy and robustness of the microphone array. In order to verify the array optimization method, two specific localization scenarios and two array optimization strategies for each localization scenario were constructed. The optimal array structure parameters were obtained through numerical iteration simulation. The localization performance of the optimal array structures obtained by the method proposed in this paper was compared with the optimal structures proposed in the literature as well as with random array structures. The simulation results show that the optimized array structure gave better positioning accuracy and robustness under both specific localization scenarios. The optimization model proposed could solve the problem of array geometric structure design based on TDOA and could achieve the customization of microphone array structures under different specific localization scenarios.

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. Multiplex immunohistochemistry staining for CD3, CD68, CD11c, PD-L1, and P40 was used to explore correlations with clinical outcomes in patients with early-stage (n=22) and locally advanced (n=36) OCSCC. These initial findings were validated through differential gene expression analysis, gene set enrichment, and immune cell deconvolution in a The Cancer Genome Atlas cohort of 163 locally advanced OCSCC tumors. Additionally, single-cell RNA sequencing (scRNA-seq) on a smaller cohort (n=10) further characterized the PD-L1 hi or PD-L1 lo cancer cells in these tumors., Results: Elevated PD-L1 expression was associated with poor outcomes in patients with locally advanced OCSCC undergoing standard adjuvant therapy, irrespective of "hot" or "cold" classification based on TILs assessment. PD-L1 hi tumors exhibited an active immune response phenotype, enriched with M1 macrophages, CD8 + T cells and T regulatory cells in the tumor microenvironment. Notably, the negative impact of PD-L1 expression on outcomes was primarily attributed to its expression by cancer cells, rather than immune cells. Furthermore, scRNA-seq revealed that immune interactions were not essential for PD-L1 upregulation in cancer cells, instead, complex regulatory networks were involved. Additionally, PD-L1 lo locally advanced tumors exhibited more complex pathway enrichment and diverse T-cell populations compared with those in the early-stage., Conclusion: Our findings underscore the prognostic significance of PD-L1 expression in locally advanced OCSCC, and unveil the complex interplay between PD-L1 expression, immune responses, and molecular pathways in the tumor microenvironment. This study provides insights that may inform future therapeutic strategies, including the possibility of tailored immunotherapeutic approaches for patients with PD-L1 hi locally advanced OCSCC., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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 STEM ) cells or dysfunctional T progenitor exhausted (T PEX ) cells. To that end, we demonstrated that T STEM 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. To address this issue, we developed a production protocol to generate T STEM -like CAR-T cells enriched for expression of genes in cell replication pathways. Compared with conventional CAR-T cells, T STEM -like CAR-T cells had enhanced proliferative capacity and increased cytokine secretion after CAR stimulation, including after chronic CAR stimulation in vitro. These responses were dependent on the presence of CD4 + T cells during T STEM -like CAR-T cell production. Adoptive transfer of T STEM -like CAR-T cells induced better control of established tumors and resistance to tumor rechallenge in preclinical models. These more favorable outcomes were associated with increased persistence of T STEM -like CAR-T cells and an increased memory T cell pool. Last, T STEM -like CAR-T cells and anti-programmed cell death protein 1 (PD-1) treatment eradicated established tumors, and this was associated with increased tumor-infiltrating CD8 + CAR + T cells producing interferon-γ. In conclusion, our CAR-T cell protocol generated T STEM -like CAR-T cells with enhanced therapeutic efficacy, resulting in increased proliferative capacity and persistence in vivo.

Background: Patients with BRAF -mutant and wild-type melanoma have different response rates to immune checkpoint blockade therapy. However, the reasons for this remain unknown. To address this issue, we investigated the precise immune composition resulting from BRAF mutation in treatment-naive melanoma to determine whether this may be a driver for different response to immunotherapy., Methods: In this study, we characterized the treatment-naive immune context in patients with BRAF -mutant and BRAF wild-type ( BRAF -wt) melanoma using data from single-cell RNA sequencing, bulk RNA sequencing, flow cytometry and immunohistochemistry (IHC)., Results: In single-cell data, BRAF -mutant melanoma displayed a significantly reduced infiltration of CD8 + T cells and macrophages but also increased B cells, natural killer (NK) cells and NKT cells. We then validated this finding using bulk RNA-seq data from the skin cutaneous melanoma cohort in The Cancer Genome Atlas and deconvoluted the data using seven different algorithms. Interestingly, BRAF -mutant tumors had more CD4 + T cells than BRAF -wt samples in both primary and metastatic cohorts. In the metastatic cohort, BRAF -mutant melanoma demonstrated more B cells but less CD8 + T cell infiltration when compared with BRAF -wt samples. In addition, we further investigated the immune cell infiltrate using flow cytometry and multiplex IHC techniques. We confirmed that BRAF -mutant melanoma metastases were enriched for CD4 + T cells and B cells and had a co-existing decrease in CD8 + T cells. Furthermore, we then identified B cells were associated with a trend for improved survival (p=0.078) in the BRAF -mutant samples and Th2 cells were associated with prolonged survival in the BRAF -wt samples., Conclusions: In conclusion, treatment-naive BRAF -mutant melanoma has a distinct immune context compared with BRAF -wt melanoma, with significantly decreased CD8 + T cells and increased B cells and CD4 + T cells in the tumor microenvironment. These findings indicate that further mechanistic studies are warranted to reveal how this difference in immune context leads to improved outcome to combination immune checkpoint blockade in BRAF -mutant melanoma., Competing Interests: Competing interests: RAS has received fees for professional services from Qbiotics, Novartis, Merck Sharp & Dohme, NeraCare, AMGEN, Bristol-Myers Squibb, Myriad Genetics, GlaxoSmithKline. GL has received professional consulting fees from Aduro Biotech, Amgen, Array Biopharma, Boehringer Ingelheim International GmbH, Bristol-Myers Squibb, Highlight Therapeutics SL, Merck Sharpe & Dohme, Novartis Pharma AG, Pierre Fabre, QBiotics Group, Regeneron Pharmaceuticals, SkylineDX BV (all unrelated to this work). PJN received research funding from Roche/Genentech, BMS, MSD, Allergan, Compugen, Crispr Therapeutics. The other authors declare no conflicts of interest., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

Chronic stimulation of CD8 + T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.

Detection of multiple lane markings on road surfaces is an important aspect of autonomous vehicles. Although a number of approaches have been proposed to detect lanes, detecting multiple lane markings, particularly across a large number of frames and under varying lighting conditions, in a consistent manner is still a challenging problem. In this paper, we propose a novel approach for detecting multiple lanes across a large number of frames and under various lighting conditions. Instead of resorting to the conventional approach of processing each frame to detect lanes, we treat the overall problem as a multitarget tracking problem across space and time using the integrated probabilistic data association filter (IPDAF) as our basis filter. We use the intensity of the pixels as an augmented feature to correctly group multiple lane markings using the Hough transform. By representing these extracted lane markings as splines, we then identify a set of control points, which becomes a set of targets to be tracked over a period of time, and thus across a large number of frames. We evaluate our approach on two different fronts, covering both model- and machine-learning-based approaches, using two different datasets, namely the Caltech and TuSimple lane detection datasets, respectively. When tested against model-based approach, the proposed approach can offer as much as 5%, 12%, and 3% improvements on the true positive, false positive, and false positives per frame rates compared to the best alternative approach, respectively. When compared against a state-of-the-art machine learning technique, particularly against a supervised learning method, the proposed approach offers 57%, 31%, 4%, and 9× improvements on the false positive, false negative, accuracy, and frame rates. Furthemore, the proposed approach retains the explainability, or in other words, the cause of actions of the proposed approach can easily be understood or explained.

Single gene defects that impair lymphocyte cytotoxicity can predispose to severe viral infection that normally remains subclinical. The classic severe presentation is hemophagocytic lymphohistiocytosis. Here, we report the case of a neonate who presented with cytomegalovirus palatal ulceration and bocavirus pneumonitis secondary to impaired cytotoxicity caused by biallelic mutations in the UNC13D gene., (© The Author(s) 2018. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The ability of cytotoxic lymphocytes (CL) to eliminate virus-infected or cancerous target cells through the granule exocytosis death pathway is critical to immune homeostasis. Congenital loss of CL function due to bi-allelic mutations in PRF1, UNC13D, STX11 , or STXBP2 leads to a potentially fatal immune dysregulation, familial haemophagocytic lymphohistiocytosis (FHL). This occurs due to the failure of CLs to release functional pore-forming protein perforin and, therefore, inability to kill the target cell. Bi-allelic mutations in partner proteins STXBP2 or STX11 impair CL cytotoxicity due to failed docking/fusion of cytotoxic secretory granules with the plasma membrane. One unique feature of STXBP2- and STX11-deficient patient CLs is that their short-term in vitro treatment with a low concentration of IL-2 partially or completely restores natural killer (NK) cell degranulation and cytotoxicity, suggesting the existence of a secondary, yet unknown, pathway for secretory granule exocytosis. In the current report, we studied NK and T-cell function in an individual with late presentation of FHL due to hypomorphic bi-allelic mutations in STXBP2 . Intriguingly, in addition to the expected alterations in the STXBP2 and STX11 proteins, we also observed a concomitant significant reduction in the expression of homologous STXBP1 protein and its partner STX1, which had never been implicated in CL function. Further analysis of human NK and T cells demonstrated a functional role for the STXBP1/STX1 axis in NK and CD8+ T-cell cytotoxicity, where it appears to be responsible for as much as 50% of their cytotoxic activity. This discovery suggests a unique and previously unappreciated interplay between STXBP/Munc proteins regulating the same essential granule exocytosis pathway.

Bi-allelic null mutations affecting UNC13D, STXBP2 , or STX11 result in defects of lymphocyte cytotoxic degranulation and commonly cause familial hemophagocytic lymphohistiocytosis (FHL) in early life. Patients with partial loss of function are increasingly being diagnosed after presenting with alternative features of this disease, or with HLH later in life. Here, we studied two sisters with lymphocyte degranulation defects secondary to compound heterozygote missense variants in UNC13D . The older sibling presented aged 11 with linear growth arrest and delayed puberty, 2 years prior to developing transient ischemic attacks secondary to neuroinflammation and hypogammaglobulinemia, but no FHL symptoms. Her geno-identical younger sister was initially asymptomatic but then presented at the same age with severe EBV-driven infectious mononucleosis, which was treated aggressively and did not progress to HLH. The sisters had similar natural killer cell degranulation; however, while cytotoxic activity was moderately reduced in the asymptomatic patient, it was completely absent in both siblings during active disease. Following allogeneic bone marrow transplantation at the age of 15, the older child has completely recovered NK cell cytotoxicity, is asymptomatic, and has experienced an exceptional compensatory growth spurt. Her younger sister was also successfully transplanted and is currently disease free. The current study reveals previously unappreciated manifestations of FHL in patients who inherited hypomorphic gene variants and also raises the important question of whether a threshold of minimum NK function can be defined that should protect a patient from serious disease manifestations such as HLH.

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., (© 2016 Federation of European Biochemical Societies.)

The production and delivery of functional perforin (PRF; PRF1 gene) by cytotoxic lymphocytes maintains immune homeostasis and tumour immune surveillance. In humans, inheritance of the common PRF1 polymorphism, p.A91V, (c.272C>T) found in 8-9% of the Caucasian population, with another mutated allele resulting in reduced PRF function or trafficking, has been shown to result in hyperinflammatory diseases and/or haematological cancers. In this study, we sought to investigate the function of p.A91V on a wild-type (WT) perforin background. We first developed an assay that distinguishes the relative levels of transcription of individual PRF1 alleles, including p.A91V. The p.A91V allele was seen to be expressed at similar levels as the WT allele in primary human natural killer (NK) cells, ruling out that allelic expression imbalance influenced their function. We then demonstrated that the p.A91V mutation results in protein misfolding and an appreciable reduction in NK-cell cytotoxicity in healthy carriers of p.A91V. We propose that this level of cytotoxic dysfunction may readily account for the predisposition to immune-mediated disease in individuals homozygous for p.A91V. Also, the fact that monoallelic mutations of PRF1 decrease NK-cell cytotoxicity should be considered in individuals presenting with the manifestations of immune deficiency states that impinge on NK-cell cytotoxicity.

Mutations in the perforin gene (PRF1) are a common cause of the fatal immune dysregulation disorder, familial hemophagocytic lymphohistiocytosis (type 2 FHL, FHL2). Here we report a female infant born with biallelic PRF1 mutations: a novel substitution, D49N, and a previously identified in-frame deletion, K285del. We assessed the effects of each mutation on the cytotoxicity of human NK cells in which the expression of endogenous perforin was ablated with miR30-based short hairpin (sh) RNAs. Both mutations were detrimental for function, thereby explaining the clinically severe presentation and rapidly fatal outcome. We demonstrate that D49N exerts its deleterious effect by generating an additional (third) N-linked glycosylation site, resulting in protein misfolding and degradation in the killer cell. Our data provide a rationale for treating some cases of type 2 familial hemophagocytic lymphohistiocytosis, based on the pharmacologic inhibition or modification of glycosylation.

Background: The aim of this study was to develop a shortened Crohn's Disease Activity Index (CDAI)., Methods: A short CDAI was developed retrospectively using patient-level data from four budesonide clinical trials to select variables from the full CDAI which best predicted health-related quality of life as measured by the Inflammatory Bowel Disease Questionnaire (IBDQ), using the multiple linear regression model. The validity, reliability, and responsiveness of the short CDAI compared to the original CDAI were determined using data from nine clinical trials of budesonide., Results: The variables selected for the short CDAI were abdominal pain, diarrhea frequency, and general well-being. In all nine studies involving 1373 patients with active and inactive CD (5863 visits), the Pearson correlation coefficients between the short CDAI scores and the original CDAI scores at baseline (r = 0.899, P < 0.001), and the score differences (r = 0.963, P < 0.001) were excellent. The short CDAI accounted for 82.4% of the variance of the original CDAI. The intraclass correlation coefficient for the short CDAI was marginally better than that for the full CDAI, and both demonstrated good reliability (r = 0.600 versus r = 0.549). In patients with active CD who remitted during follow-up, the mean short CDAI scores decreased from 247 to 97, a score difference of 150 ± 60 points (P < 0.001). In patients with stable CD who relapsed, the mean short CDAI scores increased from 109 to 244 points, a score difference of 135 ± 62 points (P < 0.001)., Conclusions: The short CDAI is a valid, reliable, and responsive tool for the measurement of CD activity., (Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.)

Introduction: Minimal hepatic encephalopathy (mHE) has been reported in up to 84 percent of cirrhotics. The natural history of mHE has not been well-described. We designed a three-year prospective cohort study to determine the prevalence and natural history of mHE among cirrhotic patients., Methods: The patient cohort comprising 62 consecutive outpatients with cirrhosis were assessed at baseline and followed-up with a repeat assessment three years later. The assessments include: (1) Neuropsychometric analysis (digit-symbol substitution test, block-design test, number-connection test A); (2) Clinical, biochemical assessment; and (3) Quality of life (QOL) assessment (abbreviated sickness impact profile)., Results: Baseline characteristics were: age 52.9 +/- 11.0 years; Child's A:B:C was 46:14:2. mHE was detected in 33.9 percent of the cohort. Older age, a higher Child-Pugh score and female gender were independently associated with mHE. mHE was associated with a poorer QOL. Follow-up assessment three years later showed that seven patients had died, while six were lost to follow-up; these patients had significantly higher baseline Child's scores. Of the remaining patients, 36/49 (73 percent) agreed to a repeat evaluation. In this group, none had mHE. QOL remained impaired despite the resolution of mHE., Conclusion: It has been shown for the first time that mHE can revert to a normal state in a significant proportion of patients with well-compensated cirrhosis.

Loss of Bid confers clonogenic survival to granzyme B-treated cells, however the exact role of Bid-induced mitochondrial damage--upstream or downstream of caspases--remains controversial. Here we show that direct cleavage of Bid by granzyme B, but not caspases, was required for granzyme B-induced apoptosis. Release of cytochrome c and SMAC, but not AIF or endonuclease G, occurred in the absence of caspase activity and correlated with the onset of apoptosis and loss of clonogenic potential. Loss of mitochondrial trans-membrane potential (DeltaPsim) was also caspase independent, however if caspase activity was blocked the mitochondria regenerated their DeltaPsim. Loss of DeltaPsim was not required for rapid granzyme B-induced apoptosis and regeneration of DeltaPsim following cytochrome c release did not confer clonogenic survival. This functional dissociation of cytochrome c and SMAC release from loss of DeltaPsim demonstrates the essential contribution of Bid upstream of caspase activation during granzyme B-induced apoptosis.

Immune surveillance by cytotoxic lymphocytes against cancer has been postulated for decades, but direct evidence for the role of cytotoxic lymphocytes in protecting against spontaneous malignancy has been lacking. As the rejection of many experimental cancers by cytotoxic T lymphocytes and natural killer cells is dependent on the pore-forming protein perforin (pfp), we examined pfp-deficient mice for increased cancer susceptibility. Here we show that pfp-deficient mice have a high incidence of malignancy in distinct lymphoid cell lineages (T, B, NKT), indicating a specific requirement for pfp in protection against lymphomagenesis. The susceptibility to lymphoma was accentuated by simultaneous lack of expression of the p53 gene, mutations in which also commonly predispose to human malignancies, including lymphoma. In contrast, the incidence and age of onset of sarcoma was unaffected in p53-deficient mice. Pfp-deficient mice were at least 1,000-fold more susceptible to these lymphomas when transplanted, compared with immunocompetent mice in which tumor rejection was controlled by CD8(+) T lymphocytes. This study is the first that implicates direct cytotoxicity by lymphocytes in regulating lymphomagenesis.

Natural tumor surveillance capabilities of the host were investigated in six different mouse tumor models where endogenous interleukin (IL)-12 does or does not dictate the efficiency of the innate immune response. Gene-targeted and lymphocyte subset-depleted mice were used to establish the relative importance of natural killer (NK) and NK1.1(+) T (NKT) cells in protection from tumor initiation and metastasis. In the models examined, CD3(-) NK cells were responsible for tumor rejection and protection from metastasis in models where control of major histocompatibility complex class I-deficient tumors was independent of IL-12. A protective role for NKT cells was only observed when tumor rejection required endogenous IL-12 activity. In particular, T cell receptor Jalpha281 gene-targeted mice confirmed a critical function for NKT cells in protection from spontaneous tumors initiated by the chemical carcinogen, methylcholanthrene. This is the first description of an antitumor function for NKT cells in the absence of exogenously administered potent stimulators such as IL-12 or alpha-galactosylceramide.

We provide the first demonstration, using experimental and spontaneous models of metastasis in C57BL/6 (B6) (RM-1 prostate carcinoma) and BALB/c (DA3 mammary carcinoma) mice, that tumor metastasis is primarily controlled by perforin-dependent cytotoxicity mediated by NK1.1+ cells. MHC class Ilow RM-1 and DA3 tumor cells were sensitive in vitro to Fas-mediated lysis or spleen NK cells in a perforin-dependent fashion. Perforin-deficient NK cells did not lyse these tumors, and perforin-deficient mice were 10-100-fold less proficient than wild-type mice in rejecting the metastasis of tumor cells to the lung. Fas ligand mutant gld mice displayed uncompromised protection against tumor metastasis. Depletion of NK subsets resulted in greater numbers of metastases than observed in perforin-deficient mice, suggesting that perforin-independent effector functions of NK cells may also contribute to protection from tumor metastasis.

Met-ase-1 is a 30 000 Mr serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3(-) large granular lymphocytes. We screened a mouse genomic library with rat Met-ase-1 cDNA, and obtained bacteriophage clones that contained the mouse Met-ase-1 gene. The mouse Met-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases. Mouse Met-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3(-) GM1(+) large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1(+) cell line 4 - 16. Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouse Met-ase-1 mRNA. The 5' flanking region of the mouse Met-ase-1 gene also shares considerable regions of identity with the 5' flanking region of the rat Met-ase-1 gene. A 3.3 kb segment of 5' sequence flanking the mouse Met-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines. The transcriptional activity of the mouse Met-ase-1 5' flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4 - 16 mouse NK1.1(+) cell line, and weak in several mouse pre-NK cell lines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouse Met-ase-1. The predicted hexapropeptide of mouse Met-ase-1 (Asn-6 to Gln-1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouse Met-ase-1 in mammalian COS-7 cells. Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouse Met-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1.

Human Met-ase-1 is a member of a family of cytotoxic lymphocyte serine proteases (granzymes), but is expressed specifically in CD3- large granular lymphocytes with natural killer cell activity. We have devised a polymerase chain reaction strategy to delete the predicted hexapropeptide of human Met-ase-1 (Ser-6 to Gln-1), to enable its expression and activation in mammalian COS cells. In addition, using peptide immunization we have derived a unique and specific monoclonal antibody detecting human Met-ase-1. Western blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the human Met-ase-1 gene encodes a serine proteinase that specifically hydrolyzes substrates containing a methionine (Met-) side chain at P1. The expression of active human Met-ase-1 and the generation of a specific anti-human Met-ase-1 monoclonal antibody will now enable a detailed structure/function analysis of key amino acids that confer this unusual serine protease specificity.

Rat natural killer cell Met-ase-1 (RNK-Met-1) is a 30,000 M(r) serine protease (granzyme) found in the cytolytic granules of CD3- large granular lymphocytes (LGL) with natural killer (NK) activity. To characterize the genomic sequences responsible for the CD3- LGL-restricted expression of this gene, we screened a rat genomic library with RNK-Met-1 cDNA, and obtained bacteriophage clones that contained the RNK-Met-1 gene. The RNK-Met-1 gene comprises 5 exons and spans approximately 5.2 kilobases (kb), exhibiting a similar structural organization to a class of CTL-serine proteases with protease catalytic residues encoded near the borders of exons 2, 3, and 5. The 5'-flanking region of the RNK-Met-1 gene contains a number of putative promoter and enhancer regulatory elements and shares several regions of homology with the 5'-flanking region of the mouse perforin gene. We have prepared nested deletions from approximately 3.3 kb of the 5'-flanking region of the RNK-Met-1 gene, and inserted these upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These 5'-flanking RNK-Met-1-CAT constructs were transiently transfected into rat LGL leukemia, T-lymphoma, and basophilic leukemia cell lines. The transcriptional activity of the RNK-Met-1 5'-flanking region was strong, restricted to the RNK-16 LGL leukemia and controlled by several positive cis-acting regions spread over at least 3.3 kb. The longest and most active 5'-flanking region (-3341 to -33) was also used to drive specific expression of beta-galactosidase in RNK-16. These data are consistent with the NK cell-specific expression of RNK-Met-1 and suggest the potential utility of this gene promoter in the development of transgene models of NK cell biology in vivo.

Upon interaction with target cells, cytotoxic T lymphocytes and natural killer cells vectorially secrete highly specialized cytoplasmic granules containing perforin and a family of serine proteases (granzymes). This granule exocytosis mechanism of cytolysis is of patho-physiological importance, and usually results in target cell DNA fragmentation. Neither perforin nor granzymes possess inherent nuclease activity, but in combination they can induce target cell apoptosis. Perforin forms transmembrane pores in the target cell, thereby enabling granzymes to access target cell substrates. The target cell substrates of granzymes are unknown, but granzyme A binding and cleavage of the nuclear shuttle protein nucleolin in target cells demonstrates that granzymes may act on nuclear substrates. Furthermore, the presence of granzyme B and other granzyme activities in the nucleus of cytotoxic lymphocytes indicates that granzymes can be transported from the cytoplasm to the nucleus. It is hypothesized that perforin enables effector granzymes to enter the target cell cytoplasm and following their transport into the nucleus, granzymes cleave specific target cell nuclear proteins to activate autolytic endonucleases that fragment DNA. In cytotoxic effectors, these nuclear substrates are normally protected from granzymes by endogenous inhibitors.

Expression of the pore-forming protein perforin is normally restricted to the cytolytic granules of cytotoxic T lymphocytes and natural killer cells. Perforin, which causes cell death by osmotic lysis, has the ability to form transmembrane channels in target cell membranes. This function makes perforin crucial in the granule-exocytosis model of T cell-mediated cytotoxicity. In the present study, variants of the mouse cytotoxic T lymphocyte cell line CTLL-R8 have been produced which express human perforin. A full-length cDNA clone (HP-10) encoding human perforin was inserted in the sense orientation into the expression plasmid pCMV5neo. The resultant construct, designated pCMV5neoHP-10, was used to transfect CTLL-R8 cells. Of eight G418-resistant clones studied, four clones expressed human perforin mRNA by Northern analysis and three of these clones also expressed human perforin protein by Western blotting. The expression of human perforin protein was associated with a pronounced (55-74%) and consistent reduction in the killing of three target cell lines, P815, YAC-1, and EL4, compared with parental CTLL-R8 cells. The reduction in target cell lysis could not be attributed to nonspecific effects of the transfection, as clones transfected with neo alone showed no reduction in killing in comparison with parental CTLL-R8 cells. Clones expressing human perforin showed very similar growth characteristics, surface phenotype, and N-alpha-benzyloxycarbonyl-l-thiobenzyl-esterase release compared with untransfected CTLL-R8 cells. The mechanism of reduction of cytolysis is unclear but may involve competition by human perforin in the handling or packaging of endogenous granule constituents (including mouse perforin) or assembly of human perforin into mouse polyperforin channels in target cell membranes. The expression of human perforin in mouse cytotoxic T cells provides a potential model for studying how cytotoxic T cells process, package, utilize, and protect themselves against the perforin molecules they produce.