A Viral Immunoevasin Controls Innate Immunity by Targeting the Prototypical Natural Killer Cell Receptor Family.

Natural killer (NK) cells play a key role in innate immunity by detecting alterations in self and non-self ligands via paired NK cell receptors (NKRs). Despite identification of numerous NKR-ligand interactions, physiological ligands for the prototypical NK1.1 orphan receptor remain elusive. Here, we identify a viral ligand for the inhibitory and activating NKR-P1 (NK1.1) receptors. This murine cytomegalovirus (MCMV)-encoded protein, m12, restrains NK cell effector function by directly engaging the inhibitory NKR-P1B receptor. However, m12 also interacts with the activating NKR-P1A/C receptors to counterbalance m12 decoy function. Structural analyses reveal that m12 sequesters a large NKR-P1 surface area via a "polar claw" mechanism. Polymorphisms in, and ablation of, the viral m12 protein and host NKR-P1B/C alleles impact NK cell responses in vivo. Thus, we identify the long-sought foreign ligand for this key immunoregulatory NKR family and reveal how it controls the evolutionary balance of immune recognition during host-pathogen interplay.

The IRE1 endoplasmic reticulum stress sensor activates natural killer cell immunity in part by regulating c-Myc.

Natural killer (NK) cells are critical mediators of host immunity to pathogens. Here, we demonstrate that the endoplasmic reticulum stress sensor inositol-requiring enzyme 1 (IRE1α) and its substrate transcription factor X-box-binding protein 1 (XBP1) drive NK cell responses against viral infection and tumors in vivo. IRE1α-XBP1 were essential for expansion of activated mouse and human NK cells and are situated downstream of the mammalian target of rapamycin signaling pathway. Transcriptome and chromatin immunoprecipitation analysis revealed c-Myc as a new and direct downstream target of XBP1 for regulation of NK cell proliferation. Genetic ablation or pharmaceutical blockade of IRE1α downregulated c-Myc, and NK cells with c-Myc haploinsufficency phenocopied IRE1α-XBP1 deficiency. c-Myc overexpression largely rescued the proliferation defect in IRE1α-/- NK cells. Like c-Myc, IRE1α-XBP1 also promotes oxidative phosphorylation in NK cells. Overall, our study identifies a IRE1α-XBP1-cMyc axis in NK cell immunity, providing insight into host protection against infection and cancer.

The Tumor Necrosis Factor Superfamily Member RANKL Suppresses Effector Cytokine Production in Group 3 Innate Lymphoid Cells.

While signals that activate group 3 innate lymphoid cells (ILC3s) have been described, the factors that negatively regulate these cells are less well understood. Here we found that the tumor necrosis factor (TNF) superfamily member receptor activator of nuclear factor κB ligand (RANKL) suppressed ILC3 activity in the intestine. Deletion of RANKL in ILC3s and T cells increased C-C motif chemokine receptor 6 (CCR6)+ ILC3 abundance and enhanced production of interleukin-17A (IL-17A) and IL-22 in response to IL-23 and during infection with the enteric murine pathogen Citrobacter rodentium. Additionally, CCR6+ ILC3s produced higher amounts of the master transcriptional regulator RORγt at steady state in the absence of RANKL. RANKL-mediated suppression was independent of T cells, and instead occurred via interactions between CCR6+ ILC3s that expressed both RANKL and its receptor, RANK. Thus, RANK-RANKL interactions between ILC3s regulate ILC3 abundance and activation, suggesting that cell clustering may control ILC3 activity.

Impact of lower concentrations of dimethyl sulfoxide on cryopreservation of autologous hematopoietic stem cells: a systematic review and meta-analysis of controlled clinical studies.

BACKGROUND AIMS: Cryopreservation of hematopoietic stem cells (HSCs) is crucial for autologous transplantation, cord blood banking and other special circumstances. Dimethyl sulfoxide (DMSO) is used most commonly for cryopreserving HSC products but can cause infusional toxicities and affect cell viability and engraftment after transplant. A systematic review of controlled studies using lower concentrations of DMSO to cryopreserve HSC products in clinical transplant studies is needed to determine the effect of reducing DMSO concentrations on post-thaw cell viability, initial engraftment and adverse effects on patient health. METHODS: All studies identified in our systematic search (to July 11, 2023) examining the use of cryopreserved peripheral blood stem cells (PBSCs) for autologous stem cell transplantation (AHCT) were included. Meta-analysis was performed to determine how varying the concentration of DMSO during cryopreservation effects post-thaw cell viability, initial engraftment and adverse effects on patient health. RESULTS: A total of 1547 studies were identified in our systematic search, with seven published articles meeting eligibility for inclusion in meta-analysis. All patients underwent AHCT using (PBSCs) to treat hematologic malignancies. The viability of CD34+ cells post thaw was greater when cryopreserved with 5% DMSO compared with 10% DMSO, with lower rates of adverse side effects in patients. DMSO concentration had minimal impact on rates of initial engraftment. Significant heterogeneity in outcome reporting was observed and the potential for bias was identified in all studies. CONCLUSIONS: Reducing the concentration of DMSO from 10% to 5% during cryopreservation of autologous PBSCs may improve cell viability and reduce DMSO-associated adverse effects in patients undergoing AHCT. Data from more studies with similar patients and standard outcome reporting are needed to increase confidence in our initial observations. PROTOCOL REGISTRATION: PROSPERO; registration number CRD42023476809 registered November 8, 2023.

Registered clinical trials investigating treatment with cell-derived extracellular vesicles: a scoping review.

BACKGROUND AIMS: Interest in cell-based therapy using extracellular vesicles (EVs) is intensifying, building upon promising preclinical research and a handful of published clinical studies. Registered clinical trials remain small, heterogeneous in design and underpowered to determine safety and efficacy on their own. A scoping review of registered studies can identify opportunities to pool data and perform meta-analysis. METHODS: Registered trials were identified by searching clinical trial databases (Clinicaltrials.gov, the World Health Organization International Clinical Trials Registry Platform and the Chinese Clinical Trial Registry) on June 10, 2022. RESULTS: Seventy-three trials were identified and included for analysis. Mesenchymal stromal cells (MSCs) were the most common cell type from which EVs were derived (49 studies, 67%). Among the 49 identified MSC-EV studies, 25 were controlled trials (51%) with a combined total of 3094 participants anticipated to receive MSC-derived EVs (2225 in controlled studies). Although EVs are being administered to treat a broad range of conditions, trials treating patients with coronavirus disease-2019 and/or acute respiratory distress syndrome were observed most commonly. Despite heterogeneity between studies, we anticipate that at least some of the studies could be combined in meaningful meta-analysis and that a combined sample size of 1000 patients would provide the ability to detect a ≥5% difference in mortality with MSC-EVs compared to controls and could be achieved by December 2023. CONCLUSIONS: This scoping review identifies potential barriers that may stall clinical translation of EV-based treatment, and our analysis calls for more standardized product characterization, use of quantifiable product quality attributes and consistent outcome reporting in future clinical trials.

Systematic review and meta-analysis of randomized controlled trials of mesenchymal stromal cells to treat coronavirus disease 2019: is it too late?

BACKGROUND AIMS: Evidence regarding the extent that mesenchymal stromal cells (MSCs) may improve clinical outcomes in patients with coronavirus disease 2019 (COVID-19) has been limited by marked inter-study heterogeneity, inconsistent product characterization and appreciable risk of bias (RoB). Given the evolution of treatment options and trajectory of the pandemic, an updated analysis of high-quality evidence from randomized controlled trials is needed for a timely and conclusive understanding of the effectiveness of MSCs. METHODS: A systematic literature search through March 30, 2022, identified all English language, full-text randomized controlled trials examining the use of MSCs in the treatment of COVID-19. RESULTS: Eight studies were identified (316 patients, 165 administered MSCs and 151 controls). Controls evolved significantly over time with a broad range of comparison treatments. All studies reported mortality at study endpoint. Random effects meta-analysis revealed that MSCs decreased relative risk of death (risk ratio, 0.63, 95% confidence interval, 0.42-0.94, P = 0.02, I2 = 14%) with no significant difference in absolute risk of death. MSCs decreased length of hospital stay and C-reactive protein levels and increased odds of clinical improvement at study endpoint compared with controls. Rates of adverse events and severe adverse events were similar between MSC and control groups. Only two (25%) studies reported all four International Society for Cell & Gene Therapy criteria for MSC characterization. Included studies had low (n = 7) or some (n = 1) concerns regarding RoB. CONCLUSIONS: MSCs may reduce risk of death in patients with severe or critical COVID-19 and improve secondary clinical outcomes. Variable outcome reporting, inconsistent product characterization and variable control group treatments remain barriers to higher-quality evidence and may constrain clinical usage. A master protocol is proposed and appears necessary for accelerated translation of higher-quality evidence for future applications of MSC therapy.

Modeling unrelated blood stem cell donor recruitment using simulated registrant cohorts: Assessment of human leukocyte antigen matching across ethnicity groups.

BACKGROUND: Human leukocyte antigen (HLA)-matched unrelated donors are not available for some patients considered for allogeneic hematopoietic cell transplantation, particularly among certain ethnic groups. Simulated recruitment modeling can inform efforts to find new matches for more patients. METHODS: Simulated recruits were generated by assigning a pair of donor HLA haplotypes from historical data files and matched against HLA data of patient searches in the Canadian Blood Services Stem Cell Registry. Recruitment cohorts reflected the proportion of five specific ethnic groups in the 2016 Canadian census data. RESULTS: Novel 8/8 HLA matches between simulated recruits and patients increased linearly with larger recruitment cohorts. The proportion of novel 8/8 HLA matches from Caucasian, Hispanic, and Native American/First Nations recruits was equal to or greater than their relative proportion in the recruited cohort (match to: recruit ratio (MRR) ≥ 1). In contrast, African American and Asian & Pacific Islander recruits represented a smaller proportion of novel matches relative to their percentage of the recruited cohort (MRR <1). The proportion of novel 7/8 HLA-matches from each ethnic group was approximately the same as their proportion in the recruited cohort (MRR ~ 1) and high rates of 7/8 HLA-matching already exist within the Canadian Blood Services registry for all ethnic groups. CONCLUSION: Continued large recruitment cohorts are needed to add new 8/8 HLA matches to registry inventories. Likelihoods of novel HLA matches varied across ethnic groups, reflecting varied HLA haplotype frequencies across groups. Simulated cohort modeling can inform recruitment strategies that will generate new donor options for patients.

HLA-haplotype redundancy and rareness in Canadian Blood Services' Stem Cell Registry and Cord Blood Bank: Novel metrics for optimizing utility.

BACKGROUND: The utility of unrelated donor registries that support allogeneic hematopoietic cell transplantation could be optimized through greater understanding of redundancy and rareness of HLA phenotypes. METHODS: HLA phenotype rareness was determined using known HLA haplotype frequencies. Donor redundancy was determined through pairwise comparison of donor HLA profiles within an inventory. RESULTS: Among 61,730 registrants in the Canadian Blood Services (CBS) Stem Cell Registry (SCR) with high resolution HLA typing at 5 loci, 6.6% of HLA phenotypes were redundant with variation across ethnic groups (8.3% of Caucasian phenotypes; 8% of Native American/First Nations, 4.4% of Asia-Pacific Islanders (API), 2.1% of Hispanic, 0.7% of African-American (AFA), and 4.5% of other ethnicities). A total of 18.5% of registrants had redundant HLA phenotypes with variation across ethnic groups. All 3716 cord blood units in the CBS's cord blood bank (CBB) had high resolution HLA typing at 5 loci and 202 units were redundant (5.4%) comprising 78 HLA phenotypes, with varying rareness. Repeated HLA phenotypes were from Caucasian donors (77%), multiple ethnicity (13%), API (9%), and AFA (1%). Registrants and CBUs with AFA ethnicity had the rarest phenotypes while Caucasian ethnicity was associated with the most common HLA phenotypes. CONCLUSIONS: Redundancy was greater in the SCR compared to the CBB and was most common with CAU ethnicity. Recruiting non-Caucasian registrants and continued cord blood banking should reduce redundancy. A sub-inventory of redundant donors and cord blood units could support new uses for donor-supported cellular therapies that do not require HLA matching.

Mesenchymal stem/stromal cell-based therapies for COVID-19: First iteration of a living systematic review and meta-analysis: MSCs and COVID-19.

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) and their secreted products are a promising therapy for COVID-19 given their immunomodulatory and tissue repair capabilities. Many small studies were launched at the onset of the pandemic, and repeated meta-analysis is critical to obtain timely and sufficient statistical power to determine efficacy. METHODS AND FINDINGS: All English-language published studies identified in our systematic search (up to February 3, 2021) examining the use of MSC-derived products to treat patients with COVID-19 were identified. Risk of bias (RoB) was assessed for all studies. Nine studies were identified (189 patients), four of which were controlled (93 patients). Three of the controlled studies reported on mortality (primary analysis) and were pooled through random-effects meta-analysis. MSCs decreased the risk of death at study endpoint compared with controls (risk ratio, 0.18; 95% confidence interval [CI], 0.04 to 0.74; P = .02; I2 = 0%), although follow-up differed. Among secondary outcomes, interleukin-6 levels were most commonly reported and were decreased compared with controls (standardized mean difference, -0.69; 95% CI, -1.15 to -0.22; P = .004; I2 = 0%) (n = 3 studies). Other outcomes were not reported consistently, and pooled estimates of effect were not performed. Substantial heterogeneity was observed between studies in terms of study design. Adherence to published ISCT criteria for MSC characterization was low. In two of nine studies, RoB analysis revealed a low to moderate risk of bias in controlled studies, and uncontrolled case series were of good (3 studies) or fair (2 studies) quality. CONCLUSION: Use of MSCs to treat COVID-19 appears promising; however, few studies were identified, and potential risk of bias was detected in all studies. More controlled studies that report uniform clinical outcomes and use MSC products that meet standard ISCT criteria should be performed. Future iterations of our systematic search should refine estimates of efficacy and clarify potential adverse effects.

Use of CRISPR/Cas9 gene editing to improve chimeric antigen-receptor T cell therapy: A systematic review and meta-analysis of preclinical studies.

BACKGROUND: Chimeric antigen-receptor T (CAR-T) cells represent great promise in cancer treatment. CRISPR/Cas9 gene editing in preclinical studies has enabled the development of enhanced CAR-T products with improved function and reduced toxicity. METHODS: A systematic review of preclinical animal studies was conducted to determine the efficacy and safety of this approach. RESULTS: 3753 records were identified (to September 9, 2020), with 11 studies using CRISPR/Cas9 gene editing in combination with CAR-T therapy against human cells in animal models of acute leukemia (four studies), glioma (two studies), melanoma (two studies), and other cancers (three studies). Compared with unedited controls, gene-edited CAR-T cells reduced tumor volume in treated animals and improved survival. No adverse side effects were reported. Use of allogeneic "third-party" CAR-T cells appears feasible. Improved efficacy was achieved through both knock-in and knockout gene editing of various targets implicated in immune function. Targeting multiple genes also appears feasible. Significant heterogeneity in study design and outcome reporting was observed, and potential bias was identified in all studies. CONCLUSION: CRISPR/Cas9 gene editing enables manufacturing of CAR-T cells with improved anti-cancer effects. Future studies should reduce unintentional bias and heterogeneity of study designs and strive to augment long-term persistence of edited cells. PROTOCOL REGISTRATION: PROSPERO; registration number CRD42020220313 registered November 30, 2020.

Demand and usage of unrelated donor products for allogeneic haematopoietic cell transplantation during the COVID-19 pandemic: A Canadian Blood Services Stem Cell Registry analysis.

BACKGROUND AND OBJECTIVES: Understanding changes in the demand and usage of unrelated allogeneic haematopoietic cell transplantation (HCT) donors during the COVID-19 pandemic is needed to optimize pandemic preparedness of registry and donor collection services. The aim of this study was to understand the extent to which the pandemic has impacted the demand and usage of unrelated donors and cord blood units (CBUs) at Canadian Blood Services (CBS). MATERIALS AND METHODS: Data regarding stem cell donor interest and product usage for unrelated allogeneic HCT were retrieved from the database at CBS using de-identified anonymous information. RESULTS: Unrelated donor searches for Canadian patients remained unchanged by the pandemic, reflecting stable demand. The number of unrelated allogeneic transplants performed within Canada also remained stable, while the number of cord blood transplants increased, chiefly for paediatric patients. Requests for donor verification typing, a first signal of potential interest, increased from domestic centres during the first 6 months of the pandemic and decreased from international centres, before returning to baseline levels. The proportion of transplants for Canadian patients who used stem cell products procured from Canadian donors increased between 3 and 6 months after the start of the pandemic before returning to baseline and appears to be increasing again more than 1 year after the start of the pandemic. Use of CBUs for Canadian paediatric patients increased and remains elevated. CONCLUSION: Demand for unrelated adult HCT donors has remained stable despite the evolving pandemic with a transient and recurring increased interest and usage of domestic adult donors. Use of CBUs for paediatric patients has increased and remains elevated. Registries and donor collection centres should maintain the capacity to expand services for domestic donor collection during pandemics to offset threats to international donor usage.

Augmentation of NK Cell Proliferation and Anti-tumor Immunity by Transgenic Expression of Receptors for EPO or TPO.

Many investigational adoptive immunotherapy regimens utilizing natural killer (NK) cells require the administration of interleukin-2 (IL-2) or IL-15, but these cytokines cause serious dose-dependent toxicities. To reduce or preclude the necessity for IL-2 use, we investigated whether genetic engineering of NK cells to express the erythropoietin (EPO) receptor (EPOR) or thrombopoietin (TPO) receptor (c-MPL) could be used as a method to improve NK cell survival and function. Viral transduction of NK-92 cells to express EPOR or c-MPL receptors conveyed signaling via appropriate pathways, protected cells from apoptosis, augmented cellular proliferation, and increased cell cytotoxic function in response to EPO or TPO ligands in vitro. In the presence of TPO, viral transduction of primary human NK cells to express c-MPL enhanced cellular proliferation and increased degranulation and cytokine production toward target cells in vitro. In contrast, transgenic expression of EPOR did not augment the proliferation of primary NK cells. In immunodeficient mice receiving TPO, in vivo persistence of primary human NK cells genetically modified to express c-MPL was higher compared with control NK cells. These data support the concept that genetic manipulation of NK cells to express hematopoietic growth factor receptors could be used as a strategy to augment NK cell proliferation and antitumor immunity.

Using umbilical cord blood for regenerative therapy: Proof or promise?

The identification of nonhematopoietic progenitor cells in cord blood has spawned great interest in using cord blood cells for new indications in regenerative therapy. Many preclinical studies demonstrated improvement in reperfusion and markers of organ recovery using cord blood-derived cells in a range of animal models. Initial results heralded increasing clinical interest regarding the use of cord blood for regenerative therapy. Initial clinical studies were largely uncontrolled feasibility studies that were case series and reported on small numbers of patients. The emergence of controlled studies has been slower, although multiple controlled studies have been conducted in patients with cerebral palsy and type I diabetes. Heterogeneity in the cellular product, patients, study design, and the timing of outcome measurements remains barriers to meta-analysis and a clearer understanding of efficacy. Controlled studies of modest size have been reported for a range of additional conditions. The conduct of controlled clinical trials to evaluate potential new uses of cord blood for regenerative therapy remains essential. None of the indications studied to date can be regarded as proven. Moreover, consistency in outcome reporting in terms of the instruments used and the time points for assessment after therapy are needed, including longer follow-up of study participants. Frequent and careful evaluation of the evidence will allow cord blood banks, health care providers, and patients to assess potential new options in the use of cord blood for regenerative therapy.

Factors associated with registrant availability for unrelated adult donor hematopoietic stem cell donation: Analysis of the stem cell registry at Canadian Blood Services.

BACKGROUND: Greater use of unrelated donors to support hematopoietic cell transplantation can be hampered by unavailability of registrants when identified as potential candidates for donation. METHODS: Multivariate analysis was performed to identify donor factors associated with availability for verification of human leukocyte antigen typing (VT) needed before donor activation. All VT requests for registrants on the Canadian Blood Services Stem Cell Registry between 1 January and 31 December 2018 were reviewed (n = 1358). RESULTS: Potential donors identified by transplant centers were categorized as available at the time of VT but ineligible for medical or other reasons (n = 130 and excluded from further analysis), available (n = 622) or unavailable (n = 566) due to scheduling, loss of interest, and/or inability to contact. With multivariate analysis, registrants who previously donated blood, those recruited online or from blood donation clinics, and a shorter interval between registration and VT request were significantly correlated with increased donor availability. Donor sex and geographic location, however, displayed no correlation. CONCLUSION: Online registration and recruitment at whole blood donation centers should be enhanced to increase the availability of registrants at VT. More insight is needed to maintain registrant availability following community in-person recruitment events, especially if the interval between registration and activation is prolonged. Recruitment of male registrants who are well informed should not negatively impact availability.

Impact of Exercise Training on Hematological Outcomes Following Hematopoietic Cell Transplantation: A Scoping Review.

PURPOSE: Hematopoietic cell transplantation (HCT) is associated with significant risk prior to hematopoietic engraftment. Endurance exercise can modify the bone marrow microenvironment, alter hematopoiesis and accelerate hematopoietic regeneration in mouse models of transplantation. METHODS: A systematic review was conducted to clarify the impact of exercise on clinically relevant hemato-logical outcomes in patients following HCT. RESULTS: A systematic search of the literature identified 13 studies (total of 615 participants; 313 in study arms). Studies included exercise regimens that were primarily low-to-moderate intensity. A total of five studies re-ported on engraftment and length of stay, which were largely unchanged with intervention. Rates of graft-ver-sus host disease were reported in six studies whereas red cell and platelet transfusion needs were reported in four studies, neither of which was different with exercise. Survival was reported in four studies and was significantly improved by exercise in one study. CONCLUSIONS: Exercise in patients receiving HCT appears feasible and safe. Heterogeneity in type and intensity of exercise was observed and few studies examined high intensity exercise. Outcome reporting was inconsis-tent regarding transplant-related outcomes. Standardized hematological outcome measures are needed to clarify the impact of higher intensity exercise on HCT.

Iron-withdrawing anti-infectives for new host-directed therapies based on iron dependence, the Achilles' heel of antibiotic-resistant microbes.

The iron dependence of antibiotic-resistant microbes represents an Achilles' heel that can be exploited broadly. The growing global problem of antibiotic resistance of microbial pathogens wherein microbes become resistant to the very antibiotics used against them during infection is linked not only to our health uses but also to agribusiness practices and the changing environment. Here we review mechanisms of microbial iron acquisition and host iron withdrawal defense, and the influence of iron withdrawal on the antimicrobial activity of antibiotics. Antibiotic-resistant microbes are unaltered in their iron requirements, but iron withdrawal from microbes enhances the activities of various antibiotics and importantly suppresses outgrowth of antibiotic-exposed resistant microbial survivors. Of the three therapeutic approaches available to exploit microbial iron susceptibility, including (1) use of gallium as a non-functional iron analogue, (2) Trojan horse conjugates of microbial siderophores carrying antibiotics, and (3) new generation iron chelators, purposely designed as anti-microbials, the latter offers various advantages. For instance, these novel anti-microbial chelators overcome the limitations of conventional clinically-used hematological chelators which display host toxicity and are not useful antimicrobials. 3-Hydroxypyridin-4-one-containing polymeric chelators appear to have the highest potential. DIBI (developmental code name) is a well-developed lead candidate, being a low molecular weight, water-soluble copolymer with enhanced iron binding characteristics, strong anti-microbial and anti-inflammatory activities, low toxicity for animals and demonstrated freedom from microbial resistance development. DIBI has been shown to enhance antibiotic efficacy for antibiotic-resistant microbes during infection, and it also prevents recovery growth and resistance development during microbe exposure to various antibiotics. Because DIBI bolsters innate iron withdrawal defenses of the infected host, it has potential to provide a host-directed anti-infective therapy.

Iron Sequestrant DIBI, a Potential Alternative for Nares Decolonization of Methicillin-Resistant Staphylococcus aureus, Is Anti-infective and Inhibitory for Mupirocin-Resistant Isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) opportunistic infections are a major health burden. Decolonization of hospitalized patients with mupirocin (MUP) has reduced the incidence of infection but has led to MUP resistance. DIBI is a developmental-stage anti-infective agent that sequesters bacterial iron and bolsters innate host iron-withdrawal defenses. Clinical isolates possessing low, high, or no MUP resistance all had similarly high susceptibilities to DIBI. Intranasal DIBI reduced nares bacterial burdens in mice to the same extent as MUP. No resistance was found after exposure to DIBI.

Mouse Cytomegalovirus m153 Protein Stabilizes Expression of the Inhibitory NKR-P1B Ligand Clr-b.

Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing stressed and infected cells through multiple germ line-encoded receptor-ligand interactions. Missing-self recognition involves NK cell sensing of the loss of host-encoded inhibitory ligands on target cells, including MHC class I (MHC-I) molecules and other MHC-I-independent ligands. Mouse cytomegalovirus (MCMV) infection promotes a rapid host-mediated loss of the inhibitory NKR-P1B ligand Clr-b (encoded by Clec2d) on infected cells. Here we provide evidence that an MCMV m145 family member, m153, functions to stabilize cell surface Clr-b during MCMV infection. Ectopic expression of m153 in fibroblasts augments Clr-b cell surface levels. Moreover, infections using m153-deficient MCMV mutants (Δm144-m158 and Δm153) show an accelerated and exacerbated Clr-b downregulation. Importantly, enhanced loss of Clr-b during Δm153 mutant infection reverts to wild-type levels upon exogenous m153 complementation in fibroblasts. While the effects of m153 on Clr-b levels are independent of Clec2d transcription, imaging experiments revealed that the m153 and Clr-b proteins only minimally colocalize within the same subcellular compartments, and tagged versions of the proteins were refractory to coimmunoprecipitation under mild-detergent conditions. Surprisingly, the Δm153 mutant possesses enhanced virulence in vivo, independent of both Clr-b and NKR-P1B, suggesting that m153 potentially targets additional host factors. Nevertheless, the present data highlight a unique mechanism by which MCMV modulates NK ligand expression.IMPORTANCE Cytomegaloviruses are betaherpesviruses that in immunocompromised individuals can lead to severe pathologies. These viruses encode various gene products that serve to evade innate immune recognition. NK cells are among the first immune cells that respond to CMV infection and use germ line-encoded NK cell receptors (NKR) to distinguish healthy from virus-infected cells. One such axis that plays a critical role in NK recognition involves the inhibitory NKR-P1B receptor, which engages the host ligand Clr-b, a molecule commonly lost on stressed cells ("missing-self"). In this study, we discovered that mouse CMV utilizes the m153 glycoprotein to circumvent host-mediated Clr-b downregulation, in order to evade NK recognition. These results highlight a novel MCMV-mediated immune evasion strategy.

Mitochondrial adaptation in human mesenchymal stem cells following ionizing radiation.

Mitochondria are highly dynamic organelles that respond rapidly to a number of stressors to regulate energy transduction, cell death signaling, and reactive oxygen species generation. We hypothesized that mitochondrial remodeling, comprising both structural and functional alterations, following ionizing radiation (IR) may underlie some of the tenets of radiobiology. Mesenchymal stem cells (MSCs) are precursors of bone marrow stroma and are altered in acute myeloid leukemia and by radiation and chemotherapy. Here, we report on changes in mitochondrial remodeling in human MSCs following X-ray IR. Mitochondrial function was significantly increased in MSCs 4 h after IR as measured by mitochondrial oxygen consumption. Consistent with this elevated functional effect, electron transport chain supercomplexes were also increased in irradiated samples. In addition, mitochondria were significantly, albeit modestly, elongated, as measured by high-throughput automated confocal imaging coupled with automated mitochondrial morphometric analyses. We also demonstrate in fibroblasts that mitochondrial remodeling is required for the adaptation of cells to IR. To determine novel mechanisms involved in mitochondrial remodeling, we performed quantitative proteomics on isolated mitochondria from cells following IR. Label-free quantitative mitochondrial proteomics revealed notable changes in proteins in irradiated samples and identified prosaposin, and potentially its daughter protein saposin-B, as a potential candidate for regulating mitochondrial function following IR. Whereas research into the biologic effects of cellular irradiation has long focused on nuclear DNA effects, our experimental work, along with that of others, is finding that mitochondrial effects may have broader implications in the field of stress adaptation and cell death in cancer (including leukemia) and other disease states.-Patten, D. A., Ouellet, M., Allan, D. S., Germain, M., Baird, S. D., Harper, M.-E., Richardson, R. B. Mitochondrial adaptation in human mesenchymal stem cells following ionizing radiation.

Willingness of volunteers from Canadian Blood Service's Stem Cell Registry to donate blood, marrow, and other tissues for regenerative therapy.

BACKGROUND: As research surrounding cell-based regenerative therapy advances toward human trials, greater demand for cell products sourced from healthy donors will arise. The extent to which volunteers in Canadian Blood Services Stem Cell Registry would be willing to donate cells to support regenerative therapy is not known and warrants exploration. METHODS: We conducted a Web-based survey to assess factors that would influence donor willingness to donate various tissues (blood, skin, fat, and bone marrow) for regenerative therapy. The survey was provided to 15,000 randomly selected donors who registered between 2013 and 2018. Data from the 1118 respondents were analyzed. RESULTS: Despite a mixed degree of familiarity with regenerative medicine, potential donors were very supportive of donating for direct patient care and for research, and increasing their familiarity by reading a brief paragraph of information on regenerative medicine increased willingness to donate. Canadian Blood Services' stem cell registrants greatly preferred supporting nonprofit groups in research and development in comparison to entities that represent profit-seeking industry involvement. The most important factors influencing donor willingness to donate were having an impact on patients, safety of donation, advancing knowledge in regenerative medicine, a manageable time commitment, and tolerable pain that could be managed. Donors were most willing to donate blood and had mixed responses to donating other tissue types. CONCLUSIONS: Adult volunteers from a national stem cell registry are willing to support donation of biospecimens for regenerative therapy.