Mitochondrial DNA levels in perfusate and bile during ex vivo normothermic machine correspond with donor liver quality.

Ex vivo normothermic machine perfusion (NMP) preserves donor organs and permits real-time assessment of allograft health, but the most effective indicators of graft viability are uncertain. Mitochondrial DNA (mtDNA), released consequent to traumatic cell injury and death, including the ischemia-reperfusion injury inherent in transplantation, may meet the need for a biomarker in this context. We describe a real time PCR-based approach to assess cell-free mtDNA during NMP as a universal biomarker of allograft quality. Measured in the perfusate fluid of 29 livers, the quantity of mtDNA correlated with metrics of donor liver health including International Normalized Ratio (INR), lactate, and warm ischemia time, and inversely correlated with inferior vena cava (IVC) flow during perfusion. Our findings endorse mtDNA as a simple and rapidly measured feature that can inform donor liver health, opening the possibility to better assess livers acquired from extended criteria donors to improve organ supply.

BioCanRx Summit for Cancer Immunotherapy 2022 Proceedings.

From 19 to 21 November 2022, BioCanRx held its first post-pandemic in-person Summit for Cancer Immunotherapy in Montreal, Canada. The meeting was well attended by patients, trainees, researchers, clinicians, and industry professionals, who came together to discuss the current state and future of biotherapeutics for cancer in Canada and beyond. Three plenaries, three keynote speakers, a lively debate, and panel discussions, together with poster sessions and a social event, made the event memorable and productive. The current state of cellular therapies, cellular engineering, clinical trials, and the role of the cancer microbiome were discussed in plenary session, and the patient voice was welcomed and present throughout the meeting, in large part due to the Learning Institute, a BioCanRx initiative to include patient partners in research. In this meeting review, we highlight the platform presentations, keynote speakers, debate combatants, panellists, and the patient perspective on the annual meeting.

Killer instincts: natural killer cells as multifactorial cancer immunotherapy.

Natural killer (NK) cells integrate heterogeneous signals for activation and inhibition using germline-encoded receptors. These receptors are stochastically co-expressed, and their concurrent engagement and signaling can adjust the sensitivity of individual cells to putative targets. Against cancers, which mutate and evolve under therapeutic and immunologic pressure, the diversity for recognition provided by NK cells may be key to comprehensive cancer control. NK cells are already being trialled as adoptive cell therapy and targets for immunotherapeutic agents. However, strategies to leverage their naturally occurring diversity and agility have not yet been developed. In this review, we discuss the receptors and signaling pathways through which signals for activation or inhibition are generated in NK cells, focusing on their roles in cancer and potential as targets for immunotherapies. Finally, we consider the impacts of receptor co-expression and the potential to engage multiple pathways of NK cell reactivity to maximize the scope and strength of antitumor activities.

Perioperative oncolytic virotherapy to counteract surgery-induced immunosuppression and improve outcomes in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a high fatality cancer with one of the worst prognoses in solid tumors. Most patients present with late stage, metastatic disease and are not eligible for potentially curative surgery. Despite complete resection, the majority of surgical patients will recur within the first two years following surgery. Postoperative immunosuppression has been described in different digestive cancers. While the underlying mechanism is not fully understood, there is compelling evidence to link surgery with disease progression and cancer metastasis in the postoperative period. However, the idea of surgery-induced immunosuppression as a facilitator of recurrence and metastatic spread has not been explored in the context of pancreatic cancer. By surveying the existing literature on surgical stress in mostly digestive cancers, we propose a novel practice-changing paradigm: alleviate surgery-induced immunosuppression and improve oncological outcome in PDAC surgical patients by administering oncolytic virotherapy in the perioperative period.

Changing liver utilization and discard rates in clinical transplantation in the ex-vivo machine preservation era.

Liver transplantation is a well-established treatment for many with end-stage liver disease. Unfortunately, the increasing organ demand has surpassed the donor supply, and approximately 30% of patients die while waiting for a suitable liver. Clinicians are often forced to consider livers of inferior quality to increase organ donation rates, but ultimately, many of those organs end up being discarded. Extensive testing in experimental animals and humans has shown that ex-vivo machine preservation allows for a more objective characterization of the graft outside the body, with particular benefit for suboptimal organs. This review focuses on the history of the implementation of ex-vivo liver machine preservation and how its enactment may modify our current concept of organ acceptability. We provide a brief overview of the major drivers of organ discard (age, ischemia time, steatosis, etc.) and how this technology may ultimately revert such a trend. We also discuss future directions for this technology, including the identification of new markers of injury and repair and the opportunity for other ex-vivo regenerative therapies. Finally, we discuss the value of this technology, considering current and future donor characteristics in the North American population that may result in a significant organ discard.

Predicting Early Graft Dysfunction and Mortality After Liver Transplant Using the De Ritis Ratio.

BACKGROUND: Predicting complications after liver transplantation (LT) remains challenging. We propose incorporating the De Ritis ratio (DRR), a widely known parameter of liver dysfunction, into current or future scoring models to predict early allograft dysfunction (EAD) and mortality after LT. METHODS: A retrospective chart review was conducted on 132 adults receiving a deceased donor LT from April 2015 to March 2020 and their matching donors. Donor variables, postoperative liver function, and DRR were correlated with the occurrence of EAD, post-transplant complications expressed by the Clavien-Dindo score, and 30-day mortality as outcome variables. RESULTS: Early allograft dysfunction was observed in 26.5% of patients and 7.6% of patients who died within 30 days after transplant. Recipients were more likely to experience EAD when receiving grafts from donation after circulatory death (P = .04), donor risk index (DRI) >2 (P = .006), ischemic injury at time-zero biopsy (P = .02), longer secondary warm ischemia time (P < .05), or higher Clavien-Dindo scores (IIIb-V; P < .001). The DRI, total bilirubin, and DRR on postoperative day 5 yielded significant associations with the primary outcomes and were used to develop the Gala-Lopez score using a weighted scoring model. This accurately predicted EAD, high Clavien-Dindo, and 30-day mortality in 75%, 81%, and 64% of patients. CONCLUSION: Including recipient and donor variables in predictive models, and for the first time DRR, as a constituent, should be regarded to predict EAD, severe complications, and 30-day mortality post-LT. Further studies will be required to validate the present findings and their applicability when using normothermic regional and machine perfusion technologies.

The Canadian Society for Immunology's 34th annual meeting 2022: symposia minireview.

The Canadian Society for Immunology 2022 Annual Meeting (June 17-20, 2022) brought together immunologists from across the country to discuss current topics and cutting-edge research in immunology. Here we highlight the published work presented during three thematic symposia (1) Immune Development and Layered Immunity; (2) Primary Immune Deficiencies from Thymic Developmental Defects to Dysregulation and Inflammation; and (3) Opposing Inflammatory and Suppressive Regulation of Anti-Tumor Immunity.

Improved overall survival in patients with high-grade serous ovarian cancer is associated with CD16a+ immunologic neighborhoods containing NK cells, T cells and macrophages.

Background: For patients with high grade serous carcinoma of the ovary (HGSC), survival rates have remained static for the last half century. Despite the presence of tumor mutations and infiltration of immune cells, existing immunotherapies have achieved little success against HGSC. These observations highlight a gap in the understanding of how the immune system functions and interacts within HGSC tumors. Methods: We analyzed duplicate core samples from 939 patients with HGSC to understand patterns of immune cell infiltration, localization, and associations with clinical features. We used high-parameter immunohistochemical/Opal multiplex, digital pathology, computational biology, and multivariate analysis to identify immune cell subsets and their associations with HGSC tumors. Results: We defined six patterns of cellular infiltration by spatially restricted unsupervised clustering of cell subsets. Each pattern was represented to some extent in most patient samples, but their specific distributions differed. Overall (OS) and progression-free survival (PFS) corresponded with higher infiltration of CD16a+ cells, and their co-localization with macrophages, T cells, NK cells, in one of six cellular neighborhoods that we defined with our spatial assessment. Conclusions: Immune cell neighborhoods containing CD16a+ cells are associated with improved OS and PFS for patients with HGSC. Patterns of immunologic neighborhoods differentiate patient outcomes, and could inform future, more precise approaches to treatment.

Preparation of mitochondrial damage-associated molecular patterns from mouse liver tissue.

Mitochondrial damage-associated molecular patterns (mitoDAMPs) are released from cells dying uncontrolled, non-apoptotic deaths, usually secondary to disease or trauma. Here, we describe preparation of mitoDAMPs from mouse liver, but this protocol can be adapted for preparation of mitoDAMPs from other species and tissues. Tissues are dissociated and then processed to isolate mitochondria. Mitochondria are then sonicated and mitoDAMPs are collected by ultracentrifugation. This procedure produces µg quantities of mitoDAMPs and facilitates research to understand their impacts in health and disease. For complete details on the use and execution of this protocol, please refer to Westhaver et al. (2022).

Manual Immunofluorescence of Formalin-Fixed Paraffin-Embedded Human Tumor Tissues.

Immunofluorescence (IF) of tumor tissues has become a key tool in the study of cancer. With a wide variety of formalin-fixed paraffin-embedded (FFPE) preserved tissues available, there are possibilities to assess large cohorts using archived tissue which may have archived associated clinical outcomes. Although best practice guidelines for the assessment of tissues have been published, a standardized method for immunofluorescence of FFPE tumor tissues is elusive. Here we provide a protocol for using classical secondary fluorescent antibodies that bind directly to the primary antibody of interest. This protocol can easily be adapted to use several primary antibodies, of different species, with unique secondary fluorophores that correspond to each species of origin. It can also be adapted for cyclic amplification-based immunofluorescence of FFPE tissues. We aim to provide a beginner-friendly and highly accessible method for immunofluorescence of FFPE-embedded tissues, hoping to enable more laboratories to take on this highly informative technique and empower them to begin IF analysis in their own tissues of interest.

Mitochondrial damage-associated molecular patterns trigger arginase-dependent lymphocyte immunoregulation.

Tissue damage leads to loss of cellular and mitochondrial membrane integrity and release of damage-associated molecular patterns, including those of mitochondrial origin (mitoDAMPs). Here, we describe the lymphocyte response to mitoDAMPs. Using primary cells from mice and human donors, we demonstrate that natural killer (NK) cells and T cells adopt regulatory phenotypes and functions in response to mitoDAMPs. NK cell-mediated cytotoxicity, interferon gamma (IFN-γ) production, T cell proliferation, and in vivo anti-viral T cell activation are all interrupted in the presence of mitoDAMPs or mitoDAMP-rich irradiated cells in in vitro and in vivo assays. Mass spectrometry analysis of mitoDAMPs demonstrates that arginase and products of its enzymatic activity are prevalent in mitoDAMP preparations. Functional validation by arginase inhibition and/or arginine add-back shows that arginine depletion is responsible for the alteration in immunologic polarity. We conclude that lymphocyte responses to mitoDAMPs reflect a highly conserved mechanism that regulates inflammation in response to tissue injury.

Natural Killer Cells: the Missing Link in Effective Treatment for High-Grade Serous Ovarian Carcinoma.

OPINION STATEMENT: Ovarian cancer (OC), especially high-grade serous cancer (HGSC), is a highly heterogeneous malignancy with limited options for curative treatment and a high frequency of relapse. Interactions between OC and the immune system may permit immunoediting and immune escape, and current standard of care therapies can influence immune cell infiltration and function within the tumor microenvironment. Natural killer (NK) cells are involved in cancer immunosurveillance and immunoediting and can be activated by therapy, but deliberate approaches to maximize NK cell reactivity for treatment of HGSC are in their infancy. NK cells may be the ideal target for immunotherapy of HGSC. The diverse functions of NK cells, and their established roles in immunosurveillance, make them attractive candidates for more precise and effective HGSC treatment. NK cells' functional capabilities differ because of variation in receptor expression and genetics, with meaningful impacts on their anticancer activity. Studying HGSC:NK cell interactions will define the features that predict the best outcomes for patients with the disease, but the highly diverse nature of HGSC will likely require combination therapies or approaches to simultaneously target multiple, co-existing features of the tumor to avoid tumor escape and relapse. We expect that the ideal therapy will enable NK cell infiltration and activity, reverse immunosuppression within the tumor microenvironment, and enable effector functions against the diverse subpopulations that comprise HGSC.

DMG26: A Targeted Sequencing Panel for Mutation Profiling to Address Gaps in the Prognostication of Multiple Myeloma.

Multiple myeloma presents with numerous primary genomic lesions that broadly dichotomize cases into hyperdiploidy or IgH translocated. Clinically, these large alterations are assessed by fluorescence in situ hybridization (FISH) for risk stratification at diagnosis. Secondary focal events, including indels and single-nucleotide variants, are also reported; however, their clinical correlates are poorly described, and FISH has insufficient resolution to assess many of them. This study examined the exonic sequences of 26 genes reported to be mutated in >1% of patients with myeloma using a custom panel. These exons were sequenced to approximately 1000 times in a cohort of 76 patients from Atlantic Canada with detailed clinical correlates and in four multiple myeloma cell lines. Across the 76 patients, 255 mutations and 33 focal copy number variations were identified. High-severity mutations and mutations predicted by FATHMM-XF to be pathogenic identified patients with significantly reduced progression-free survival. These mutations were mutually exclusive from the Revised International Staging System high-risk FISH markers and were independent of all biochemical parameters of the Revised International Staging System. Applying our panel to patients classified by FISH to be standard risk successfully reclassified patients into high- and standard-risk groups. Furthermore, three patients in our cohort each had two high-risk markers; two of these patients developed plasma cell leukemia, a rare and severe clinical sequela of multiple myeloma.

NK cell infiltration is associated with improved overall survival in solid cancers: A systematic review and meta-analysis.

The immune landscape of a tumor is highly connected to patient prognosis and response to treatment, but little is known about how natural killer (NK) cells predict overall survival (OS) among patients with solid tumors. We present the first meta-analysis on NK cell infiltration into solid tumors as a prognostic indicator for OS, considering cancer types independently, and together. Samples were collected from 1973 to 2016 with results published between 1989 and 2020. From 53 studies, we found that NK cell infiltration corresponds with decreased risk of death (HR=0.34, 95% CI: 0.26-0.46; p<0.0001). Among studies that investigated the prognostic potential of NK cells in specific regions of the tumor, intraepithelial infiltration was better predictive of OS than NK infiltration in the tumor-adjacent stroma. Generally, NK cell infiltration is lower in advanced-stage and lower-grade tumors; nevertheless, it remains prognostically beneficial. This meta-analysis highlights an important prognostic role of NK cells in solid tumors, but exposes that few studies have considered the contributions of NK cells. Toward NK cell-based immunotherapies, it will be important to understand the conditions under which NK cells can be effective agents of tumor control.

Highly efficient serum-free manipulation of miRNA in human NK cells without loss of viability or phenotypic alterations is accomplished with TransIT-TKO.

Natural killer (NK) cells are innate lymphocytes with functions that include target cell killing, inflammation and regulation. NK cells integrate incoming activating and inhibitory signals through an array of germline-encoded receptors to gauge the health of neighbouring cells. The reactive potential of NK cells is influenced by microRNA (miRNA), small non-coding sequences that interfere with mRNA expression. miRNAs are highly conserved between species, and a single miRNA can have hundreds to thousands of targets and influence entire cellular programs. Two miRNA species, miR-155-5p and miR-146a-5p are known to be important in controlling NK cell function, but research to best understand the impacts of miRNA species within NK cells has been bottlenecked by a lack of techniques for altering miRNA concentrations efficiently and without off-target effects. Here, we describe a non-viral and straightforward approach for increasing or decreasing expression of miRNA in primary human NK cells. We achieve >90% transfection efficiency without off-target impacts on NK cell viability, education, phenotype or function. This opens the opportunity to study and manipulate NK cell miRNA profiles and their impacts on NK cellular programs which may influence outcomes of cancer, inflammation and autoimmunity.

The Evolutionary Arms Race between Virus and NK Cells: Diversity Enables Population-Level Virus Control.

Viruses and natural killer (NK) cells have a long co-evolutionary history, evidenced by patterns of specific NK gene frequencies in those susceptible or resistant to infections. The killer immunoglobulin-like receptors (KIR) and their human leukocyte antigen (HLA) ligands together form the most polymorphic receptor-ligand partnership in the human genome and govern the process of NK cell education. The KIR and HLA genes segregate independently, thus creating an array of reactive potentials within and between the NK cell repertoires of individuals. In this review, we discuss the interplay between NK cell education and adaptation with virus infection, with a special focus on three viruses for which the NK cell response is often studied: human immunodeficiency virus (HIV), hepatitis C virus (HCV) and human cytomegalovirus (HCMV). Through this lens, we highlight the complex co-evolution of viruses and NK cells, and their impact on viral control.

Publisher Correction: Novel multiplex PCR-SSP method for centromeric KIR allele discrimination.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Naturally Killing the Silent Killer: NK Cell-Based Immunotherapy for Ovarian Cancer.

Ovarian cancer (OC) is diagnosed in ~22,000 women in the US each year and kills 14,000 of them. Often, patients are not diagnosed until the later stages of disease, when treatment options are limited, highlighting the urgent need for new and improved therapies for precise cancer control. An individual's immune function and interaction with tumor cells can be prognostic of the response to cancer treatment. Current emerging therapies for OC include immunotherapies, which use antibodies or drive T cell-mediated cancer recognition and elimination. In OC, these have been limited by adverse side effects and tumor characteristics including inter- and intra-tumoral heterogeneity, lack of targetable antigens, loss of tumor human leukocyte antigen expression, high levels of immunosuppressive factors, and insufficient immune cell trafficking. Natural killer (NK) cells may be ideal as primary or collateral effectors to these nascent immunotherapies. NK cells exhibit multiple functions that combat immune escape and tumor relapse: they kill targets and elicit inflammation through antigen-independent pathways and detect loss of HLA as a signal for activation. NK cells are efficient mediators of tumor immune surveillance and control, suppressed by the tumor microenvironment and rescued by immune checkpoint blockade. NK cells are regulated by a variety of activating and inhibitory receptors and already known to be central effectors across an array of existing therapies. In this article, we highlight interactions between NK cells and OC and their potential to change the immunosuppressive tumor microenvironment and participate in durable immune control of OC.

Novel Approach to Cell Surface Discrimination Between KIR2DL1 Subtypes and KIR2DS1 Identifies Hierarchies in NK Repertoire, Education, and Tolerance.

Cumulative activating and inhibitory receptor signaling controls the functional output of individual natural killer (NK) cells. Investigation of how competing signals impact response, however, has been hampered by the lack of available antibodies capable of distinguishing inhibitory and activating receptors with highly homologous ectodomains. Utilizing a novel combination of monoclonal antibodies with specificity for discrete inhibitory KIR2DL1 and activating KIR2DS1 allotypes found among 230 healthy donors, we investigated allele-specific receptor expression and function driven by KIR2DL1 and KIR2DS1 alleles. We found that co-expression of the HLA-C2 ligand diminishes KIR2DL1, but not KIR2DS1, cell surface staining, but does not impact the respective frequencies of KIR2DL1- and KIR2DS1-expressing cells within the NK repertoire. We can distinguish by flow cytometry NK cell populations expressing the most common KIR2DL1-C245 allotypes from those expressing the most common KIR2DL1-R245 allotypes, and we show that the informative differential binding anti-KIR2DL1/S1 clone 1127B is determined by amino acid residue T154. Although both KIR2DL1-C245 and KIR2DL1-R245 subtypes can be co-expressed in the same cell, NK cells preferentially express one or the other. Cells expressing KIR2DL1-C245 exhibited a lower KIR2DL1 cell surface density and lower missing-self reactivity in comparison to cells expressing KIR2DL1-R245. We found no difference, however, in sensitivity to inhibition or cell surface stability between the two KIR2DL1 isoforms, and both demonstrated similar expansion among NKG2C+ KIR2DL1+ NK cells in HCMV-seropositive healthy individuals. In addition to cell surface density of KIR2DL1, copy number of cognate HLA-C2 hierarchically impacted the effector capacity of both KIR2DL1+ cells and the tolerization of KIR2DS1+ NK cells. HLA-C2 tolerization of KIR2DS1+ NK cells could be overridden, however, by education via co-expressed self-specific inhibitory receptors, such as the heterodimer CD94/NKG2A. Our results demonstrate that effector function of NK cells expressing KIR2DL1 or KIR2DS1 is highly influenced by genetic variability and is calibrated by co-expression of additional NK receptors and cognate HLA-C2 ligands. These findings define the molecular conditions under which NK cells are activated or inhibited, potentially informing selection of donors for adoptive NK therapies.

Novel multiplex PCR-SSP method for centromeric KIR allele discrimination.

Allelic diversity of the KIR2DL receptors drive differential expression and ligand-binding affinities that impact natural killer cell function and patient outcomes for diverse cancers. We have developed a global intermediate resolution amplification-refractory mutation system (ARMS) PCR-SSP method for distinguishing functionally relevant subgroups of the KIR2DL receptors, as defined by phylogenetic study of the protein sequences. Use of the ARMS design makes the method reliable and usable as a kit, with all reactions utilizing the same conditions. Six reactions define six subgroups of KIR2DL1; four reactions define three subgroups of KIR2DL2; and five reactions define four subgroups of KIR2DL3. Using KIR allele data from a cohort of 426 European-Americans, we identified the most common KIR2DL subtypes and developed the high-throughput PCR-based methodology, which was validated on a separate cohort of 260 healthy donors. Linkage disequilibrium analysis between the different KIR2DL alleles revealed that seven allelic combinations represent more than 95% of the observed population genotypes for KIR2DL1/L2/L3. In summary, our findings enable rapid typing of the most common KIR2DL receptor subtypes, allowing more accurate prediction of co-inheritance and providing a useful tool for the discrimination of observed differences in surface expression and effector function among NK cells exhibiting disparate KIR2DL allotypes.