Out-of-Sequence Signal 3 Paralyzes Primary CD4(+) T-Cell-Dependent Immunity.

Primary T cell activation involves the integration of three distinct signals delivered in sequence: (1) antigen recognition, (2) costimulation, and (3) cytokine-mediated differentiation and expansion. Strong immunostimulatory events such as immunotherapy or infection induce profound cytokine release causing "bystander" T cell activation, thereby increasing the potential for autoreactivity and need for control. We show that during strong stimulation, a profound suppression of primary CD4(+) T-cell-mediated immune responses ensued and was observed across preclinical models and patients undergoing high-dose interleukin-2 (IL-2) therapy. This suppression targeted naive CD4(+) but not CD8(+) T cells and was mediated through transient suppressor of cytokine signaling-3 (SOCS3) inhibition of the STAT5b transcription factor signaling pathway. These events resulted in complete paralysis of primary CD4(+) T cell activation, affecting memory generation and induction of autoimmunity as well as impaired viral clearance. These data highlight the critical regulation of naive CD4(+) T cells during inflammatory conditions.

Association of dexmedetomidine use with haemodynamics, postoperative recovery, and cost in paediatric anaesthesia: a hospital registry study.

BACKGROUND: Dexmedetomidine utilisation in paediatric patients is increasing. We hypothesised that intraoperative use of dexmedetomidine in children is associated with longer postanaesthesia care unit length of stay, higher healthcare costs, and side-effects. METHODS: We analysed data from paediatric patients (aged 0-12 yr) between 2016 and 2021 in the Bronx, NY, USA. We matched our cohort with the Healthcare Cost and Utilization Project-Kids' Inpatient Database (HCUP-KID). RESULTS: Among 18 104 paediatric patients, intraoperative dexmedetomidine utilisation increased from 51.7% to 85.7% between 2016 and 2021 (P<0.001). Dexmedetomidine was dose-dependently associated with a longer postanaesthesia care unit length of stay (adjusted absolute difference [ADadj] 19.7 min; 95% confidence interval [CI]: 18.0-21.4 min; P<0.001, median length of stay of 122 vs 98 min). The association was magnified in children aged ≤2 yr undergoing short (≤60 min) ambulatory procedures (ADadj 33.3 min; 95% CI: 26.3-40.7 min; P<0.001; P-for-interaction <0.001). Dexmedetomidine was associated with higher total hospital costs of USD 1311 (95% CI: USD 835-1800), higher odds of intraoperative mean arterial blood pressure below 55 mm Hg (adjusted odds ratio [ORadj] 1.27; 95% CI: 1.16-1.39; P<0.001), and higher odds of heart rate below 100 beats min-1 (ORadj 1.32; 95% CI: 1.21-1.45; P<0.001), with no preventive effects on emergence delirium requiring postanaesthesia i.v. sedatives (ORadj 1.67; 95% CI: 1.04-2.68; P=0.034). CONCLUSIONS: Intraoperative use of dexmedetomidine is associated with unwarranted haemodynamic effects, longer postanaesthesia care unit length of stay, and higher costs, without preventive effects on emergence delirium.

Development and validation of a score for prediction of postoperative respiratory complications in infants and children (SPORC-C).

BACKGROUND: In infants and children, postoperative respiratory complications are leading causes of perioperative morbidity, mortality, and increased healthcare utilisation. We aimed to develop a novel score for prediction of postoperative respiratory complications in paediatric patients (SPORC for children). METHODS: We analysed data from paediatric patients (≤12 yr) undergoing surgery in New York and Boston, USA for score development and external validation. The primary outcome was postoperative respiratory complications within 30 days after surgery, defined as respiratory infection, respiratory failure, aspiration pneumonitis, pneumothorax, pleural effusion, bronchospasm, laryngospasm, and reintubation. Data from Children's Hospital at Montefiore were used to create the score by stepwise backwards elimination using multivariate logistic regression. External validation was conducted using a separate cohort of children who underwent surgery at Massachusetts General Hospital for Children. RESULTS: The study included data from children undergoing 32,187‬ surgical procedures, where 768 (2.4%) children experienced postoperative respiratory complications. The final score consisted of 11 predictors, and showed discriminatory ability in development, internal, and external validation cohorts with areas under the receiver operating characteristic curve of 0.85 (95% confidence interval: 0.83-0.87), 0.84 (0.80-0.87), and 0.83 (0.80-0.86), respectively. CONCLUSION: SPORC is a novel validated score for predicting the likelihood of postoperative respiratory complications in children that can be used to predict postoperative respiratory complications in infants and children.

Loss of CASK Accelerates Heart Failure Development.

[Figure: see text].

Evaluation of arterial stiffness and serum endocan levels in patients with primary aldosteronism with new-onset hypertension and long-term hypertension.

PURPOSE: There is growing evidence that prolonged exposure to high serum aldosterone concentrations results in target organ damage to the heart, kidney, and arterial wall, and that primary aldosteronism (PA) is associated with increased cardiovascular risk. In this study, we aimed to evaluate cardiovascular disease (CVD) risk indicators such as arterial stiffness [with pulse wave velocity (PWV) measurement] in PA patients and endocan levels, which is a biomarker of endothelial dysfunction. METHODS: 28 patients with PA were included in our study. As the control group, 14 patients with essential hypertension (EHT) and 28 normotensive healthy volunteers were included. Height, weight, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), serum fasting glucose, insulin, hemoglobin A1c (HbA1c), C-reactive protein (CRP), lipids and endocan levels of all subjects in the PA, EHT and control groups were measured. PWV measurements were performed to assess arterial stiffness. RESULTS: In the PA group, PWV levels were similar to the EHT group, and endocan levels were lower than the EHT group. In the PA group, PWV levels were higher than the control group, and endocan levels were lower than the control group. When we compared the PA group with new-onset HT with the PA group with long-term HT, PWV levels were higher in the PA group with long-term HT. When we compared the long-term HT group with the EHT group, PWV levels were higher in the long-term HT PA group and endocan levels were higher in the EHT group. When we compared the PA group with long-term HT with the control group, PWV levels were higher in the PA group with long-term HT, and endocan levels were similar in both groups. CONCLUSIONS: In our study, it was determined that arterial stiffness increased in PA cases with long-term HT compared to PA cases with new-onset HT, EHT cases and normotensive healthy cases. We found that endocan levels in PA patients were also lower than both EHT patients and healthy controls.

The relationship between social achievement goals and self-esteem, depression and anxiety among medical school students.

Background: Social achievement goals such as the desire to receive positive feedback from the social environment or avoid negative feedback are situations that affect an individual's quality of life and predispose them to mental disorders. Aim: The aim of this study is to investigate the relationship between social achievement goals and self-esteem, depression, and anxiety in medical school students. Materials and Methods: 400 participants, 201 of whom were female volunteers, between the ages of 18-30, studying at the Faculty of Medicine were evaluated. Sociodemographic Data Form, Social Achievement Goal Orientation Scale, Beck Anxiety Inventory, Beck Depression Inventory, and Rosenberg Self-Esteem Inventory Sub-Scale were applied to the participants. Results: A negative correlation between social development goals and depression (rs = -0.218, P < 0.001) and anxiety (rs = -0.188, P < 0.001), and a positive correlation with self-esteem (P = 0.002) were found. A statistically significant and positive correlation between social performance-avoidance goals and depression (rs = 0.233, P < 0.001) and anxiety (rs = 0.245, P < 0.001), and still statistically significant, and negative relationship with self-esteem (P = 0.001) were found. While social performance-approach goals were positively correlated with anxiety (rs = 0.192, P < 0.001) and depression (rs = 0.108, P = 0.03), no statistically significant correlation was found with self-esteem (P = 0.129). Conclusion: It has been seen that our study generally supports the other studies in the literature concerning the relations between social achievement goal subgroups and self-esteem, depression, and anxiety in university students. It will be possible to contribute to the findings with studies encompassing university students from different cities and departments and studies with a large number of participants other than students.

Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation.

ProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co-incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA-negative mutant in A549 epithelial cells and THP-1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin-like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1 ' and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination.

GRANULYSIN PEPTIDE AND GENE POLYMORPHISM IN THE PATHOGENESIS OF HASHIMOTO THYROIDITIS.

Background: Hashimoto thyroiditis (HT) is an autoimmune disease and the most common cause of hypothyroidism. The widespread lymphocyte infiltration in the thyroid gland and intolerance of the body against its thyroid antigens leads to the destruction of thyroid cells and impaired thyroid function. Granulysin (GNLY) is a cytolytic antimicrobial peptide that has been associated with a wide range of diseases such as various infections, cancer, transplantation, and skin problems. However, there are a few studies investigating the relationship between HT and granulysin. Aim: Our study aims to investigate whether granulysin levels and GNLY gene polymorphism contribute to the damaged immune response leading to HT. Material and Methods: 100 unrelated patients diagnosed with HT and 140 healthy individuals were included in our study. Frequencies of GNLY rs10180391 and rs7908 gene polymorphisms were determined using PCR- RFLP method and serum granulysin levels were determined using ELISA. Results: There is no statistical significance between patient and control groups in terms of genotype and allele frequencies of GNLY gene polymorphisms and serum levels of granulysin. Conclusion: In conclusion, granulysin and GNLY gene polymorphisms do not appear to relate to HT disease.

NEW BIOMARKERS TO PREDICT CARDIOVASCULAR RISK IN PATIENTS WITH ADRENAL INCIDENTALOMA; IRISIN AND NESFATIN-1.

Objective: In our study, we aimed to investigate the levels of irisin, nesfatin-1 and the relationship between levels of these relatively new molecules with cardiometabolic risk markers; carotid intima-media thickness (CIMT), epicardial adipose tissue (EAT) thickness in patients with nonfunctional adrenal incidentaloma (NFAI). Materials and Methods: Patients with NFAI (n=59) and age, sex and body mass index-matched healthy control subjects (n=59) were enrolled in this study. Serum glucose, insulin, C-reactive protein (CRP), lipid, irisin and nesfatin-1 levels and echocardiographic CIMT and EAT thickness measurements were performed in patients and controls. Results: The irisin level was 17.58 ± 4.38 pg/mL in the NFAI group, significantly higher (p<0.001) than 14.03 ± 4.03 pg/mL in the control group. Nesfatin-1 level was significantly lower in the NFAI group 194.98 ± 119.15 pg/mL ((p < 0.001)) versus 303.48 ± 200.78 pg/mL in the control group. A positive correlation was found between irisin and nesfatin-1 levels and CIMT and EAT thickness in the NFAI group. Conclusions: In our study, we found that irisin level was higher and nesfatin-1 level was lower in patients with NFAI, and both irisin and nesfatin-1 levels were associated with CIMT and EAT thickness in NFAI patients.

Evaluation of retinal microvascular changes in patients with prediabetes.

AIM: Diabetic retinopathy is a chronic progressive complication with neuronal cell and retinal microvascular involvement and is closely associated with blood sugar and blood pressure levels. Studies have shown that retinal neural dysfunction takes place before the microvascular changes in patients with Type 2 diabetes mellitus. The aim of this study is to compare the retinal microvascular changes of patients who are at the prediabetes stage and healthy volunteers. METHOD: Our study included 41 patients with prediabetes who were referred to the internal medicine outpatient clinic and 47 healthy volunteers. All patients underwent ophthalmologic examinations, including best visual acuity, intraocular pressure measurement, slit-lamp examination, and dilated fundus examination. Refractive error measurements were performed with the same automatic refractor-keratometer device. Typically, 3 × 3 mm macular images centered on foveola were obtained by using XR Avanti Optical Coherence Tomography Angiography with AngioVue (RTVue XR AVANTI, Optovue, Fremont, CA, USA) device. In the statistical analysis of the measurements, it was examined by Kolmogorov Smirnov test. Conditions expressed as IFG or IGT are considered as prediabetes; IFG is defined as fasting blood sugar to be between 100 and 125 mg/dL, while IGT is the condition in which the second hour value of the oral glucose tolerance test is 140-199 mg/dL. RESULTS: There was no statistically significant difference between the control and pre-DM groups in terms of mean age. The distribution of males and females between groups was statistically similar (P = 0.087). In the pre-DM group, 24 (58.6%) patients had IFG, 16 (39.0%) had IFG + IGT, and 1 (2.4%) had IGT. There were no statistically significant differences between the groups for the nonflow area (NFA) and the foveal avascular zone (FAZ) area (P > 0.05). The mean values of superficial and deep capillary plexus (DCP) density were not statistically significant differences between the groups. No statistically significant difference was found between the control group and pre-DM group in terms of the mean measurements of clinical ocular findings (P > 0.05). Retinal thicknesses were also not statistically significant differences between the groups (P > 0.05). CONCLUSION: All of the retinal measurements of both patients with prediabetes and healthy volunteers are similar. We did not find any difference between prediabetes and control groups. The ophthalmologic examinations which contain best-visual acuity, intraocular pressure measurement, slit-lamp examination, and dilated fundus examination are similar.

SnoopLigase Catalyzes Peptide-Peptide Locking and Enables Solid-Phase Conjugate Isolation.

Simple, efficient reactions for connecting biological building-blocks open up many new possibilities. Here we have designed SnoopLigase, a protein that catalyzes site-specific transamidation, forming an isopeptide bond with more than 95% efficiency between two peptide tags, SnoopTagJr and DogTag. We initially developed these components by three-part splitting of the Streptococcus pneumoniae adhesin RrgA. The units were then engineered, guided by structure, bioinformatic analysis of sequence homology, and computational prediction of stability. After engineering, SnoopLigase demonstrated high-yield coupling under a wide range of buffers and temperatures. SnoopTagJr and DogTag were functional at the N- or C-terminus, while DogTag was also functional at internal sites in proteins. Having directed reaction of SnoopTagJr and DogTag, SnoopLigase remained stably bound to the ligated product, thus reconstituting the parent domain. Separating products from unreacted starting material and catalyst is often as challenging as reactions themselves. However, solid-phase immobilization of SnoopLigase enabled the ligated SnoopTagJr-DogTag product to be eluted with high purity, free from SnoopLigase or unligated substrates. The solid-phase catalyst could then be reused multiple times. In search of a generic route to improve the resilience of enzymes, we fused SnoopTagJr to the N-terminus and DogTag to the C-terminus of model enzymes, allowing cyclization via SnoopLigase. While wild-type phytase and ß-lactamase irreversibly aggregated upon heating, cyclization using SnoopLigase conferred exceptional thermoresilience, with both enzymes retaining solubility and activity following heat treatment up to 100 °C. SnoopLigase should create new opportunities for conjugation and nanoassembly, while illustrating how to harness product inhibition and extend catalyst utility.

Dual Plug-and-Display Synthetic Assembly Using Orthogonal Reactive Proteins for Twin Antigen Immunization.

Engineering modular platforms to control biomolecular architecture can advance both the understanding and the manipulation of biological systems. Icosahedral particles uniformly displaying single antigens stimulate potent immune activation and have been successful in various licensed vaccines. However, it remains challenging to display multiple antigens on a single particle and to induce broader immunity protective across strains or even against distinct diseases. Here, we design a dually addressable synthetic nanoparticle by engineering the multimerizing coiled-coil IMX313 and two orthogonally reactive split proteins. SpyCatcher protein forms an isopeptide bond with SpyTag peptide through spontaneous amidation. SnoopCatcher forms an isopeptide bond with SnoopTag peptide through transamidation. SpyCatcher-IMX-SnoopCatcher provides a modular platform, whereby SpyTag-antigen and SnoopTag-antigen can be multimerized on opposite faces of the particle simply upon mixing. We demonstrate efficient derivatization of the platform with model proteins and complex pathogen-derived antigens. SpyCatcher-IMX-SnoopCatcher was expressed in Escherichia coli and was resilient to lyophilization or extreme temperatures. For the next generation of malaria vaccines, blocking the transmission of the parasite from human to mosquito is an important goal. SpyCatcher-IMX-SnoopCatcher multimerization of the leading transmission-blocking antigens Pfs25 and Pfs28 greatly enhanced the antibody response to both antigens in comparison to the monomeric proteins. This dual plug-and-display architecture should help to accelerate vaccine development for malaria and other diseases.

Stent luxation: Possible complication of subadventitial stenting in coronary chronic total occlusion revascularization.

The entry to the subadventitial space is not a mode of failure, but the road to success in many chronic total occlusion (CTO) percutaneous coronary intervention (PCI) cases. Long-term clinical outcomes of subadventitial stenting are favorable and similar to intraluminal stenting. However, the subadventitial space histology and physiology remains different to the coronary true intraluminal space. We report a complication specific to stenting of the subadventitial space, where overlapping stents dislocated from one another, which resulted in late non-occlusive stent thrombosis. We describe, for the first time in the literature, this complication, possible reasons behind, and the ways to avoid this potentially major mischief. © 2016 Wiley Periodicals, Inc.

Fatigue in systemic lupus erythematosus : Association with disease activity, quality of life and psychosocial factors.

OBJECTIVE: The aim of the study was to determine which disease-related factors and non-disease features can explain the presence of systemic lupus erythematosus (SLE)-related fatigue in Turkish patients. METHODS: This cross-sectional study was carried out with 99 SLE patients and 71 healthy controls. To assess fatigue and health-related quality of life (HRQoL) the participants were asked to complete two questionnaires: the short form-36 health survey (SF-36) and the multidimensional assessment of fatigue (MAF) scale. Anxiety and depression of participants were assessed by the hospital anxiety and depression scale (HADS). RESULTS: A total of 99 patients (female/male 95/4) and 71 controls (female/male 40/31) were studied. The mean age and standard deviation (±SD) of patients and controls were 43.3 ± 12.2 years and 43.2 ± 12.1 years, respectively. The mean (SD) disease duration was 7.8 ± 5.3 years and median SLE disease activity index (SLEDAI) score was 0 (range = 0-16). The level of fatigue was higher in patients compared to controls with mean MAF scores of 24.7 ± 12.2 and 12.8 ± 9.9 (p < 0.001), respectively. The HADS-D and HADS-A scores were also significantly higher in SLE patients (6.6 ± 4.3 vs. 3.6 ± 2.9, p < 0.001 and 7.2 ± 4 vs. 4.9 ± 4, p = 0.007, respectively). There were no significant associations between the MAF and SLEDAI scores (r = 0.05, p = 0.63) but MAF scores positively correlated with age, HADS-A and HADS-D scores and negatively correlated with physical component summary (PCS), mental component summary (MCS) and each domain of SF-36 except role emotional in SLE patients. CONCLUSION: Fatigue is an important factor influencing patient daily life independent from disease activity in our study. The SLE patients with severe fatigue should also be assessed for other possible underlying causes such as anxiety, depression and poor quality of life.

Dual effects of testosterone in Behcet's disease: implications for a role in disease pathogenesis.

Behcet's disease (BD) exhibits more severe disease course and higher mortality among male patients. However, underlying mechanisms of gender differences in clinical manifestations and disease severity are unclear. The aim of this study was to determine whether testosterone (T) has any role on BD pathogenesis. We studied peripheral blood mononuclear cells (PBMC) and neutrophils of BD patients and controls. Functional assay of neutrophils, cytokine measurements of culture supernatants and gene expressions on both cells were analyzed before and after T incubation. Neutrophils were significantly activated after incubation with T in only BD patients. Incubation with T caused significantly elevated interleukin (IL)-12 and IL-2 in BD. Gene expression of IL-10 was significantly downregulated after incubation with T in BD, especially in male patients. The same difference was observed in IL-10 levels in culture supernatant after T. Baseline TLR4 expression was significantly higher in BD patients compared to healthy donors (HC). Toll-like receptor (TLR) 4 expression on PBMC was significantly elevated in female BD patients. ERAP1 expressions of all patients and controls were decreased under the T effect but it differed significantly between BD vs HC. Baseline IL23R expression was higher in BD males compared with females but the difference disappeared after T. When BD patients were analyzed separately, baseline C-C motif chemokine receptor1 (CCR1), STAT4, TLR4 and KLRC4 expressions were lower in males. Despite immunosuppressive behavior in healthy subjects, T causes neutrophil hyperactivation and TH1 type immune alterations in BD patients. Our results suggest that T may have a role in BD pathogenesis by altering the expression level of IL-10, TLR4, ERAP1, CCR1.

FKBPs in bacterial infections.

BACKGROUND: FK506-binding proteins (FKBPs) contain a domain with peptidyl-prolyl-cis/trans-isomerase (PPIase) activity and bind the immunosuppressive drugs FK506 and rapamycin. FKBPs belong to the immunophilin family and are found in eukaryotes and bacteria. SCOPE OF REVIEW: In this review we describe two major groups of bacterial virulence-associated FKBPs, the trigger factor and Mip-like PPIases. Moreover, we discuss the contribution of host FKBPs in bacterial infection processes. MAJOR CONCLUSIONS: Since PPIases are regarded as alternative antiinfective drug targets we highlight current research strategies utilizing pipecolinic acid and cycloheximide derivatives as well as substrate based inhibitors. GENERAL SIGNIFICANCE: The current research strategies suggest a beneficial synergism of drug development and basic research. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Legionella-protozoa-nematode interactions in aquatic biofilms and influence of Mip on Caenorhabditis elegans colonization.

Legionella pneumophila, the causative agent of Legionnaires disease, is naturally found in aquatic habitats. The intracellular life cycle within protozoa pre-adapted the "accidental" human pathogen to also infect human professional phagocytes like alveolar macrophages. Previous studies employing the model organism Caenorhabditis elegans suggest that also nematodes might serve as a natural host for L. pneumophila. Here, we report for the first time from a natural co-habitation of L. pneumophila and environmental nematode species within biofilms of a warm water spring. In addition, we identified the protozoan species Oxytricha bifaria, Stylonychia mytilus, Ciliophrya sp. which have never been described as potential interaction partners of L. pneumophila before. Modeling and dissection of the Legionella-protozoa-nematode interaction revealed that C. elegans ruptures Legionella-infected amoebal cells and by this means incorporate the pathogen. Further infection studies revealed that the macrophage infectivity potentiator (Mip) protein of L. pneumophila, which is known to bind collagen IV during human lung infection, promotes the colonization of the intestinal tract of L4 larvae of C. elegans and negatively influences the life span of the worms. The Mip-negative L. pneumophila mutant exhibited a 32-fold reduced colonization rate of the nematodes after 48h when compared to the wild-type strain. Taken together, these studies suggest that nematodes may serve as natural hosts for L. pneumophila, promote their persistence and dissemination in the environment, and co-evolutionarily pre-adapt the pathogen for interactions with extracellular constituents of human lung tissue.

Increased antitumor effects using IL-2 with anti-TGF-ß reveals competition between mouse NK and CD8 T cells.

Because of increasing interest in the removal of immunosuppressive pathways in cancer, the combination of IL-2 with Abs to neutralize TGF-ß, a potent immunosuppressive cytokine, was assessed. Combination immunotherapy resulted in significantly greater antitumor effects. These were correlated with significant increases in the numbers and functionality of NK cells, NK cell progenitors, and activated CD8 T cells, resulting in the observed antitumor effects. Combination immunotherapy also was accompanied by lesser toxicities than was IL-2 therapy alone. Additionally, we observed a dual competition between NK cells and activated CD8 T cells such that, after immunotherapy, the depletion of either effector population resulted in the increased total expansion of the other population and compensatory antitumor effects. This study demonstrates the efficacy of this combination immunotherapeutic regimen as a promising cancer therapy and illustrates the existence of potent competitive regulatory pathways between NK cells and CD8 T cells in response to systemic activation.

Murine natural killer cell licensing and regulation by T regulatory cells in viral responses.

Natural killer (NK) cells show differential functionality based on their capability of binding to self-MHC consistent with licensing. Here we show in vivo confirmation of the physiologic effects of licensing with differential effects of NK subsets on anti-murine cytomegalovirus (anti-MCMV) responses after syngeneic hematopoietic stem cell transplantation (HSCT) or regulatory T-cell (Treg) depletion. After HSCT, depletion of licensed NK cells led to far greater viral loads in target organs early after infection compared with nondepleted and unlicensed depleted mice. There was a preferential expansion of licensed, C-type lectin-like activating receptor Ly49H+ NK cells with increased IFNγ production after infection in nondepleted mice post-HSCT and after Treg depletion. Adoptive transfer of licensed NK subsets into immunodeficient hosts provided significantly greater MCMV resistance compared with transfer of total NK populations or unlicensed subsets. In non-HSCT mice, only concurrent depletion of Tregs or TGF-ß neutralization resulted in detection of NK licensing effects. This suggests that licensed NK cells are the initial and rapidly responding population of NK cells to MCMV infection, but are highly regulated by Tregs and TGF-ß.