Differences in pulmonary function measured by oscillometry between horses with mild-moderate equine asthma and healthy controls.

BACKGROUND: The diagnosis of mild-moderate equine asthma (MEA) can be confirmed by airway endoscopy, bronchoalveolar lavage fluid (BALf) cytology, and lung function evaluation by indirect pleural pressure measurement. Oscillometry is a promising pulmonary function test method, but its ability to detect subclinical airway obstruction has been questioned. OBJECTIVES: To evaluate the differences in lung function measured by oscillometry between healthy and MEA-affected horses. STUDY DESIGN: Prospective case-control clinical study. METHODS: Thirty-seven horses were divided into healthy and MEA groups, based on history and clinical score; the diagnosis of MEA was confirmed by airway endoscopy and BALf cytology. Horses underwent oscillometry at frequencies ranging from 2 to 6 Hz. Obtained parameters included whole-breath, inspiratory, expiratory, and the difference between inspiratory and expiratory resistance (Rrs) and reactance (Xrs). Differences between oscillometry parameters at different frequencies were evaluated within and between groups by repeated-measures two-way ANOVA and post hoc tests with Bonferroni correction. Frequency dependence was compared between groups by t test. For significant parameters, a receiver operating characteristics curve was designed, cut-off values were identified and their sensitivity and specificity were calculated. Statistical significance was set at p < 0.05. RESULTS: No significant differences in Xrs and Rrs were observed between groups. The frequency dependence of whole-breath and inspiratory Xrs significantly differed between healthy (respectively, -0.03 ± 0.02 and -0.05 ± 0.02 cmH2O/L/s) and MEA (-0.1 ± 0.03 and -0.2 ± 0.02 cmH2O/L/s) groups (p < 0.05 and p < 0.01). For inspiratory Xrs frequency dependence, a cut-off value of -0.06 cmH2O/L/s was identified, with 86.4% (95% CI: 66.7%-95.3%) sensitivity and 66.7% (95% CI: 41.7%-84.8%) specificity. MAIN LIMITATIONS: Sample size, no BALf cytology in some healthy horses. CONCLUSIONS: Oscillometry can represent a useful non-invasive tool for the diagnosis of MEA. Specifically, the evaluation of the frequency dependence of Xrs may be of special interest.

Towards personalized medicine for the treatment of equine asthma.

Although horses with asthma share similar clinical signs, the heterogeneity of the disease in terms of severity, triggering factors, inflammatory profile, and pathological features has hindered our ability to define biologically distinct subgroups. The recognition of phenotypes and endotypes could enable the development of precision medicine, including personalized, targeted therapy, to benefit affected horses. While in its infancy in horses, this review outlines the phenotypes of equine asthma and discusses how knowledge gained from targeted therapy in human medicine can be applied to evaluate the potential opportunities for personalized medicine in equine asthma and to suggest avenues for research to advance this emerging field.

Comparative study of the bronchodilator efficacy and adverse effects of salbutamol and hyoscine butylbromide in horses with severe asthma.

BACKGROUND: Salbutamol and hyoscine butylbromide (HBB) are commonly used bronchodilators in horses with severe asthma (SA). OBJECTIVE: To compare the bronchodilation potency, duration, and adverse effects of salbutamol and HBB in SA. ANIMALS: Six horses in exacerbation of SA. METHODS: The effects of inhaled salbutamol (1000 µg) and HBB (150 mg, IV) were compared in a randomized, blinded, crossover experiment. Lung function, intestinal borborygmi and heart rate were assessed before and sequentially until 180 minutes after drug administration, and analyzed with 2-way repeated-measures ANOVA and Dunnett's multiple comparison tests. RESULTS: Both treatments caused a similar improvement in lung function. Pulmonary resistance and reactance returned to baseline values within 30 minutes after HBB administration, whereas salbutamol improved reactance until 180 minutes (mean improvement at 180 minutes of 0.040 Kpa/L/s, 95% CI = 0.004 to 0.076; P = .02 for salbutamol and of 0.009 Kpa/L/s, 95% CI = -0.028 to 0.045; P = .98 for HBB for the resistance at 3 Hz and of 0.040 Kpa/L/s, 95% CI = 0.007 to 0.074; P = .01 for salbutamol and of 0.009 Kpa/L/s, 95% CI = -0.024 to 0.042; P = .97 for HBB for the reactance at 7 Hz). From 5 to 30 minutes after HBB administration, the heart rate accelerated (mean increase of 3.3 beats per minute, 95% CI = -6.6 to 13.1; P = .92 for salbutamol, and of 13.0 beats per minute, 95% CI = 3.6 to 22.4; P = .002 for HBB at 30 minutes) and the gut sounds decreased (mean reduction of 1.3, 95% CI = -0.1 to 2.8; P = .09 for salbutamol and of 2.8 for the gastrointestinal auscultation score, 95% CI = 1.4 to 4.3; P < .0001 for HBB at 30 minutes). CONCLUSIONS AND CLINICAL IMPORTANCE: Both drugs have a similar bronchodilator potency but with a longer duration for salbutamol. Gastrointestinal and cardiovascular effects were noted only with HBB, suggesting the preferential use of salbutamol to relieve bronchoconstriction in horses with asthma.

Glutathione Supplementation Prevents Neonatal Parenteral Nutrition-Induced Short- and Long-Term Epigenetic and Transcriptional Disruptions of Hepatic H2O2 Metabolism in Guinea Pigs.

The parenteral nutrition (PN) received by premature newborns is contaminated with peroxides that induce global DNA hypermethylation via oxidative stress. Exposure to peroxides could be an important factor in the induction of chronic diseases such as those observed in adults who were born preterm. As endogenous H2O2 is a major regulator of glucose-lipid metabolism, our hypothesis was that early exposure to PN induces permanent epigenetic changes in H2O2 metabolism. Three-day-old guinea pigs were fed orally (ON), PN or glutathione-enriched PN (PN+GSSG). GSSG promotes endogenous peroxide detoxification. After 4 days, half the animals were sacrificed, and the other half were fed ON until 16 weeks of age. The liver was harvested. DNA methylation and mRNA levels were determined for the SOD2, GPx1, GCLC, GSase, Nrf2 and Keap1 genes. PN induced GPx1 hypermethylation and decreased GPx1, GCLC and GSase mRNA. These findings were not observed in PN+GSSG. PN+GSSG induced Nrf2 hypomethylation and increased Nrf2 and SOD2 mRNA. These observations were independent of age. In conclusion, in neonatal guinea pigs, PN induces epigenetic changes, affecting the expression of H2O2 metabolism genes. These changes persist for at least 15 weeks after PN. This disruption may signify a permanent reduction in the capacity to detoxify peroxides.

Pharmacological evidence that the inhibitory effects of prostaglandin E2 are mediated by the EP2 and EP4 receptors in human neutrophils.

Prostaglandin E2 (PGE2) is a recognized inhibitor of granulocyte functions. However, most of the data supporting this was obtained when available pharmacological tools mainly targeted the EP2 receptor. Herein, we revisited the inhibitory effect of PGE2 on reactive oxygen species production, leukotriene biosynthesis, and migration in human neutrophils. Our data confirm the inhibitory effect of PGE2 on these functions and unravel that the effect of PGE2 on human neutrophils is obtained by the combined action of EP2 and EP4 agonism. Accordingly, we also demonstrate that the inhibitory effect of PGE2 is fully prevented only by the combination of EP2 and EP4 receptor antagonists, underscoring the importance of targeting both receptors in the effect of PGE2. Conversely, we also show that the inhibition of ROS production by human eosinophils only involves the EP4 receptor, despite the fact that they also express the EP2 receptor.

Low levels of microRNA-21 in neutrophil-derived exosomes may contribute to airway smooth muscle hyperproliferation in horses with severe asthma.

OBJECTIVE: Neutrophilic inflammation is associated with the degree of airway obstruction in severe equine asthma (SEA), but the contribution of these leukocytes to bronchial remodeling remains ill defined. Neutrophils could cause structural alterations of the airways by the release of exosomes, a type of cell-derived nanoparticles that can modify the biology of local and distant cells. Neutrophil-derived exosomes have been shown to increase airway smooth muscle (ASM) cell proliferation in humans and horses. Therefore, this study aimed to identify neutrophil exosomal microRNAs (miRs) implicated in the regulation of ASM biology in SEA. ANIMALS: 6 horses with SEA and 6 healthy controls. METHODS: The expression of selected miRs in exosomes from peripheral neutrophils was studied by quantitative PCR. The effects of miR-21 transfection in ASM cells were evaluated by gene expression analysis and proliferation studies. RESULTS: The miR-21 was downregulated in neutrophil exosomes from SEA horses, and it attenuated the proliferation of ASM cells stimulated with lipopolysaccharide. CLINICAL RELEVANCE: The lower level of miR-21 in neutrophil-derived exosomes could contribute to ASM hyperproliferation, which could, in turn, promote the thickening of the bronchial wall in SEA.

Prediction of plasma ctDNA fraction and prognostic implications of liquid biopsy in advanced prostate cancer.

No consensus strategies exist for prognosticating metastatic castration-resistant prostate cancer (mCRPC). Circulating tumor DNA fraction (ctDNA%) is increasingly reported by commercial and laboratory tests but its utility for risk stratification is unclear. Here, we intersect ctDNA%, treatment outcomes, and clinical characteristics across 738 plasma samples from 491 male mCRPC patients from two randomized multicentre phase II trials and a prospective province-wide blood biobanking program. ctDNA% correlates with serum and radiographic metrics of disease burden and is highest in patients with liver metastases. ctDNA% strongly predicts overall survival, progression-free survival, and treatment response independent of therapeutic context and outperformed established prognostic clinical factors. Recognizing that ctDNA-based biomarker genotyping is limited by low ctDNA% in some patients, we leverage the relationship between clinical prognostic factors and ctDNA% to develop a clinically-interpretable machine-learning tool that predicts whether a patient has sufficient ctDNA% for informative ctDNA genotyping (available online: https://www.ctDNA.org ). Our results affirm ctDNA% as an actionable tool for patient risk stratification and provide a practical framework for optimized biomarker testing.

Encapsulated Streptococcus suis impairs optimal neutrophil functions which are not rescued by priming with colony-stimulating factors.

The porcine pathogen and zoonotic agent Streptococcus suis induces an exacerbated inflammation in the infected hosts that leads to sepsis, meningitis, and sudden death. Several virulence factors were described for S. suis of which the capsular polysaccharide (CPS) conceals it from the immune system, and the suilysin exhibits cytotoxic activity. Although neutrophils are recruited rapidly upon S. suis infection, their microbicidal functions appear to be poorly activated against the bacteria. However, during disease, the inflammatory environment could promote neutrophil activation as mediators such as the granulocyte colony-stimulating factor granulocyte (G-CSF) and the granulocyte-macrophages colony-stimulating factor (GM-CSF) prime neutrophils and enhance their responsiveness to bacterial detection. Thus, we hypothesized that CPS and suilysin prevent an efficient activation of neutrophils by S. suis, but that G-CSF and GM-CSF rescue neutrophil activation, leading to S. suis elimination. We evaluated the functions of porcine neutrophils in vitro in response to S. suis and investigated the role of the CPS and suilysin on cell activation using isogenic mutants of the bacteria. We also studied the influence of G-CSF and GM-CSF on neutrophil response to S. suis by priming the cells with recombinant proteins. Our study confirmed that CPS prevents S. suis-induced activation of most neutrophil functions but participates in the release of neutrophil-extracellular traps (NETs). Priming with G-CSF did not influence cell activation, but GM-CSF strongly promote IL-8 release, indicating its involvement in immunomodulation. However, priming did not enhance microbicidal functions. Studying the interaction between S. suis and neutrophils-first responders in host defense-remains fundamental to understand the immunopathogenesis of the infection and to develop therapeutical strategies related to neutrophils' defense against this bacterium.

Breath characteristics and adventitious lung sounds in healthy and asthmatic horses.

BACKGROUND: Standard thoracic auscultation suffers from limitations, and no systematic analysis of breath sounds in asthmatic horses exists. OBJECTIVES: First, characterize breath sounds in horses recorded using a novel digital auscultation device (DAD). Second, use DAD to compare breath variables and occurrence of adventitious sounds in healthy and asthmatic horses. ANIMALS: Twelve healthy control horses (ctl), 12 horses with mild to moderate asthma (mEA), 10 horses with severe asthma (sEA) (5 in remission [sEA-], and 5 in exacerbation [sEA+]). METHODS: Prospective multicenter case-control study. Horses were categorized based on the horse owner-assessed respiratory signs index. Each horse was digitally auscultated in 11 locations simultaneously for 1 hour. One-hundred breaths per recording were randomly selected, blindly categorized, and statistically analyzed. RESULTS: Digital auscultation allowed breath sound characterization and scoring in horses. Wheezes, crackles, rattles, and breath intensity were significantly more frequent, higher (P < .001, P < .01, P = .01, P < .01, respectively) in sEA+ (68.6%, 66.1%, 17.7%, 97.9%, respectively), but not in sEA- (0%, 0.7%, 1.3%, 5.6%) or mEA (0%, 1.0%, 2.4%, 1.7%) horses, compared to ctl (0%, 0.6%, 1.8%, -9.4%, respectively). Regression analysis suggested breath duration and intensity as explanatory variables for groups, wheezes for tracheal mucus score, and breath intensity and wheezes for the 23-point weighted clinical score (WCS23). CONCLUSIONS AND CLINICAL IMPORTANCE: The DAD permitted characterization and quantification of breath variables, which demonstrated increased adventitious sounds in sEA+. Analysis of a larger sample is needed to determine differences among ctl, mEA, and sEA- horses.

Respiratory oscillometry testing in relation to exercise in healthy and asthmatic Thoroughbreds.

BACKGROUND: Racehorses may experience exercise-induced bronchodilation or bronchoconstriction, with potential differences between healthy and asthmatic individuals. OBJECTIVES: To identify exercise-related lung function variations by oscillometry in racehorses, compare lung function between healthy and mild equine asthma (MEA) horses, assess oscillometry's potential as a predictor of racing fitness. STUDY DESIGN: Prospective case-control clinical study. METHODS: Fourteen Thoroughbred racehorses (5 healthy, 9 MEA) underwent a protocol including respiratory oscillometry at rest, exercise with fitness monitoring, oscillometry at 15 and 45 min post-exercise, and bronchoalveolar lavage fluid (BALf) cytology. Oscillometry parameters (resistance [Rrs] and reactance [Xrs]) were compared within and between healthy and MEA groups at different timepoints. Associations between Rrs and Xrs at rest and 15 min post-exercise and BALf cytology and fitness indices were evaluated. RESULTS: MEA horses showed higher Rrs at 15 min post-exercise (0.6 ± 0.2 cmH2 O/L/s) than healthy horses (0.3 ± 0.1 cmH2 O/L/s) (p < 0.01). In healthy horses, Rrs decreased at 15 min post-exercise compared with resting values (0.5 ± 0.1 cmH2 O/L/s) (p = 0.04). In MEA horses, oscillometry parameters did not vary with time. Post-exercise Xrs inversely correlated with total haemosiderin score (p < 0.01, r2 = 0.51). Resting Rrs inversely correlated with speed at 200 bpm (p = 0.03, r2 = -0.61), and Xrs with maximum heart rate (HR) during exercise (p = 0.02, r2 = -0.62). Post-exercise Rrs inversely correlated with mean (p = 0.04, r2 = -0.60) and maximum speed (p = 0.04, r2 = -0.60), and HR variability (p < 0.01, r2 = -0.74). MAIN LIMITATIONS: Small sample size, oscillometry repeatability not assessed, potential interference of upper airway obstructions, external variables influencing fitness indices. CONCLUSIONS: Oscillometry identified lung function differences between healthy and MEA horses at 15 min post-exercise. Only healthy horses exhibited exercise-induced bronchodilation. Oscillometry showed potential in predicting subclinical airway obstruction.

Severe asthma in horses is associated with increased airway innervation.

BACKGROUND: Altered innervation structure and function contribute to airway hyperresponsiveness in human asthma, yet the role of innervation in airflow limitation in asthma in horses remains unknown. HYPOTHESIS: To characterize peribronchial innervation in horses with asthma. We hypothesized that airway innervation increases in horses with asthma compared with controls. ANIMALS: Formalin-fixed lung samples from 8 horses with severe asthma and 8 healthy horses from the Equine Respiratory Tissue Biobank. Ante-mortem lung function was recorded. METHODS: Blinded case-control study. Immunohistochemistry was performed using rabbit anti-s100 antibody as a neuronal marker for myelinating and non-myelinating Schwann cells. The number and cumulative area of nerves in the peribronchial region and associated with airway smooth muscle were recorded using histomorphometry and corrected for airway size. RESULTS: Both the number (median [IQR]: 1.87 × 10-5 nerves/µm2 [1.28 × 10-5 ]) and the cumulative nerve area (CNA; 1.03 × 10-3 CNA/µm2 [1.57 × 10-3 ]) were higher in the peribronchial region of horses with asthma compared with controls (5.17 × 10-6 nerves/µm2 [3.76 × 10-6 ], 4.14 × 10-4 CNA/µm2 [2.54 × 10-4 ], Mann-Whitney, P = .01). The number of nerves within or lining airway smooth muscle was significantly higher in horses with asthma (4.47 × 10-6 nerves/µm2 [5.75 × 10-6 ]) compared with controls (2.26 × 10-6 nerves/µm2 [1.16 × 10-6 ], Mann-Whitney, P = .03). CONCLUSIONS AND CLINICAL IMPORTANCE: Asthma in horses is associated with greater airway innervation, possibly contributing to airway smooth muscle remodeling and exacerbating severity of the disease.

Aspect elucidation of a physicochemical pretreatment for continuous decrystallization.

The purpose of this study is to understand the operating conditions of a physicochemical pretreatment process for lignocellulosic biomass using homogeneous acid catalysts. Four parameters were studied: moisture content, acid catalyst, type of biomass and reactor morphology. The different types of biomass (perennial grasses: sugarcane bagasse, corn stover; flowering plants: cannabis (stalks and leaves); hardwoods (pulp and bark): poplar, sugar maple; softwood bark) were processed in a meat grinder with sulfuric acid. Furthermore, softwood bark was used to change the moisture content, acid catalyst and reactor morphology. Biomass moisture above 17 wt% yielded less than 50 wt% glucose. Sulfuric acid, by far, had the best performance with a 74.5 wt% glucose yield in the meat grinder. The glucose yield showed a direct relationship with the non-carbohydrate components of biomass (lignin, ash, etc).

Disturbances of the Lung Glutathione System in Adult Guinea Pigs Following Neonatal Vitamin C or Cysteine Deficiency.

In premature infants receiving parenteral nutrition, oxidative stress is a trigger for the development of bronchopulmonary dysplasia, which is an important factor in the development of adult lung diseases. Neonatal vitamin C and glutathione deficiency is suspected to induce permanent modification of redox metabolism favoring the development of neonatal and adult lung diseases. A total of 64 3-day-old guinea pigs were fed an oral diet that was either complete or deficient in vitamin C (VCD), cysteine (CD) (glutathione-limiting substrate) or both (DD) for 4 days. At 1 week of age, half of the animals were sacrificed while the other started a complete diet until 12 weeks of age. At 1 week, the decrease in lung GSH in all deficient groups was partially explained by the oxidation of liver methionine-adenosyltransferase. mRNA levels of kelch-like ECH-associated protein 1 (Keap1), glutathione-reductase (Gsr) and glutaredoxin-1 (Glrx) were significantly lower only in CD but not in DD. At 12 weeks, glutathione levels were increased in VCD and CD. Keap1, Gsr and Glrx mRNA were increased, while glutathione-reductase and glutaredoxin proteins were lower in CD, favoring a higher glutathionylation status. Both neonatal deficiencies result in a long-term change in glutathione metabolism that could contribute to lung diseases' development.

Parenteral Cysteine Supplementation in Preterm Infants: One Size Does Not Fit All.

Due to their gastrointestinal immaturity or the severity of their pathology, many neonates require parenteral nutrition (PN). An amino acid (AA) solution is an important part of PN. Cysteine is a key AA for protein and taurine synthesis, as well as for glutathione synthesis, which is a cornerstone of antioxidant defenses. As cysteine could be synthesized from methionine, it is considered a nonessential AA. However, many studies suggest that cysteine is a conditionally essential AA in preterm infants due to limitations in their capacity for cysteine synthesis from methionine and the immaturity of their cellular cysteine uptake. This critical review discusses the endogenous synthesis of cysteine, its main biological functions and whether cysteine is a conditionally essential AA. The clinical evidence evaluating the effectiveness of the current methods of cysteine supplementation, between 1967 and 2023, is then reviewed. The current understanding of cysteine metabolism is applied to explain why these methods were not proven effective. To respond to the urgent need for changing the current methods of parenteral cysteine supplementation, glutathione addition to PN is presented as an innovative alternative with promising results in an animal model. At the end of this review, future directions for research in this field are proposed.

Neutrophil Extracellular Vesicles and Airway Smooth Muscle Proliferation in the Natural Model of Severe Asthma in Horses.

Extracellular vesicles (EVs) contribute to intercellular communication through the transfer of their rich cargo to recipient cells. The EVs produced by LPS-stimulated neutrophils from healthy humans and horses increase airway smooth muscle (ASM) proliferation, but the roles of neutrophil EVs in asthma are largely unexplored. The aim of this study was to determine whether neutrophil-derived EVs isolated during the remission or exacerbation of asthma influence ASM proliferation differentially. Peripheral blood neutrophils were collected during remission and exacerbation in eight horses affected by severe asthma. The cells were cultured (±LPS), and their EVs were isolated by ultracentrifugation and characterized by laser scattering microscopy and proteomic analysis. The proliferation of ASM co-incubated with EVs was monitored in real time by electrical impedance. Two proteins were significantly upregulated during disease exacerbation in neutrophil EVs (MAST4 and Lrch4), while LPS stimulation greatly altered the proteomic profile. Those changes involved the upregulation of neutrophil degranulation products, including proteases known to induce myocyte proliferation. In agreement with the proteomic results, EVs from LPS-stimulated neutrophils increased ASM proliferation, without an effect of the disease status. The inhalation of environmental LPS could contribute to asthma pathogenesis by activating neutrophils and leading to ASM hyperplasia.

Dose-Response Effects of Glutathione Supplement in Parenteral Nutrition on Pulmonary Oxidative Stress and Alveolarization in Newborn Guinea Pig.

In premature infants, glutathione deficiency impairs the capacity to detoxify the peroxides resulting from O2 metabolism and those contaminating the parenteral nutrition (PN) leading to increased oxidative stress, which is a major contributor to bronchopulmonary dysplasia (BPD) development. In animals, the supplementation of PN with glutathione prevented the induction of pulmonary oxidative stress and hypoalveolarization (characteristic of BPD). Hypothesis: the dose of glutathione that corrects the plasma glutathione deficiency is sufficient to prevent oxidative stress and preserve pulmonary integrity. Three-day-old guinea pigs received a PN, supplemented or not with GSSG (up to 1300 µg/kg/d), the stable form of glutathione in PN. Animals with no handling other than being orally fed constituted the control group. After 4 days, lungs were removed to determine the GSH, GSSG, redox potential and the alveolarization index. Total plasma glutathione was quantified. The effective dose to improve pulmonary GSH and prevent the loss of alveoli was 330 µg/kg/d. A 750 µg/kg/d dose corrected the low-plasma glutathione, high-pulmonary GSSG and oxidized redox potential. Therefore, the results suggest that, in a clinical setting, the dose that improves low-plasma glutathione could be effective in preventing BPD development.

Biosynthesis and metabolism of endocannabinoids and their congeners from the monoacylglycerol and N-acyl-ethanolamine families.

The endocannabinoids 2-arachidonoyl-glycerol (2-AG) and N-arachidonoyl-ethanolamine (AEA) are eicosanoids implicated in numerous physiological processes like appetite, adipogenesis, inflammatory pain and inflammation. They mediate most of their physiological effects by activating the cannabinoid (CB) receptors 1 and 2. Other than directly binding to the CB receptors, 2-AG and AEA are also metabolized by most eicosanoid biosynthetic enzymes, yielding many metabolites that are part of the oxyendocannabinoidome. Some of these metabolites have been found in vivo, have the ability to modulate specific receptors and thus potentially influence physiological processes. In this review, we discuss the biosynthesis and metabolism of 2-AG and AEA, as well as their congeners from the monoacyl-glycerol and N-acyl-ethanolamine families, with a special focus on the metabolism by oxygenases involved in arachidonic acid metabolism. We highlight the knowledge gaps in our understanding of the regulation and roles the oxyendocannabinoidome mediators.

Comprehensive immune profiling of patients with advanced urothelial or renal cell carcinoma receiving immune checkpoint blockade.

Immune checkpoint inhibitors (ICI) are used in the treatment of urothelial and renal cell cancers. While some patients may have exceptional responses, better predictive biomarkers are needed. We profiled the circulating immune compartment of patients receiving ICI to identify possible immune markers associated with immunotherapy response or resistance. Peripheral blood samples were collected prior to, and 3 weeks after initiation of ICI. Using mass cytometry, 26 distinct immune populations were identified. Responders to immune checkpoint inhibitors had higher frequencies of naïve CD4+ T-cells, and lower frequencies of CD161+ Th17 cells and CCR4+ Th2 cells. Non-responders had a higher frequency of circulating PD1+ T-cells at baseline; there was a subsequent decrease in frequency with exposure to ICI with a concomitant increase in Ki67 expression. Flow cytometry for cytokines and chemokine receptors showed that CD4+ T cells of non-responder patients expressed less CXCR4 and CCR7. In addition, their PD1- CD4+ T cells had higher TNFα and higher CCR4 expression, while their PD1+ CD4+ T cells had higher interferon γ and lower CCR4 expression. The role of γ/δ T-cells was also explored. In responders, these cells had higher levels of interferon γ, TNFα and CCR5. One patient with a complete response had markedly higher frequency of γ/δ T-cells at baseline, and an expansion of these cells after treatment. This case was analyzed using single-cell gene expression profiling. The bulk of the γ/δ T cells consisted of a single clone of Vγ9/Vδ2 cells both before and after expansion, although the expansion was polyclonal. Gene expression analysis showed that exposure to an ICI led to a more activated phenotype of the γ/δ T cells. In this study, the circulating immune compartment was shown to have potential for biomarker discovery. Its dynamic changes during treatment may be used to assess response before radiographic changes are apparent, and these changes may help us delineate mechanisms that underpin both response and resistance to ICI. It also hypothesizes a potential role for γ/δ T cells as effector cells in some cases.