Immature Neutrophils Research Articles

Stability and comparison of complete blood count parameters between capillary and venous blood samples.

This study assessed the comparability of complete blood count (CBC) parameters between capillary and venous samples, and extended previous research by examining the influence of different storage temperatures on CBC stability up to 7 days after sample collection. Venous and capillary blood samples were collected from 93 adult patients. Hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean platelet volume (MPV), leukocytes, lymphocytes, basophils, eosinophils, erythrocytes, red cell distribution width (RDW), immature granulocytes (IG), immature reticulocyte fraction (IRF), monocytes, neutrophils, platelets, and reticulocytes were measured. Deming regression and mean relative differences between venous and capillary measurements were contrasted with desirable total allowable error (TEa). Stability was assessed in 20-27 venous blood samples stored at 4, 21-22, or 30°C, and analyzed at 0, 24, 48, 72, 96, 120, 144, and 168 h. Mean relative change with respect to baseline measurements was compared to the desirable TEa to determine acceptable stability. Deming regression demonstrated strong linear correlations and acceptable variation between venous and capillary measurements. Erythrocytes, Hb, Ht, MCH, MCV, RDW, reticulocytes, and platelets showed acceptable stability for at least 96 h at 4°C. Mean relative change exceeded desirable TEa after 24 h at 30°C for all parameters, except erythrocytes, Hb, leukocytes, and MCH. Clinical laboratory specialists and clinicians should be aware of potential differences between venous and capillary measurements, and the influence of storage conditions. Clinical validity of delayed CBC analysis depends on the clinical situation and required precision of the result.

Identification of immunosuppressive and tumor-promoting neutrophils in bone marrow of human and human immune system mice

Abstract Neutrophils in cancer patients were identified as a specialized heterogeneous population that suppress anti-tumor responses and accelerate tumor progression. However, the developmental stage and the source of this population remain elusive. To address this issue, we defined human neutrophil development based on previous publications and demonstrated that immature neutrophils predominate tumor tissues of patients. On the quest to identify the origin of immature neutrophils, we unexpectedly discovered that immature neutrophils collected from bone marrow of healthy people and human immune system (HIS) mice are naturally immunosuppressive and tumor promoting, whereas the mouse counterpart is not. To further investigate the linkage between peripheral and bone marrow immature neutrophils, we created a NCG-Gfi1 −/−strain, which overcomes the issue of poor human neutrophil engraftment in periphery. NCG-Gfi1 −/−HIS mice manifest similar kinetics of immature neutrophil expansion as cancer patients both in bone marrow and periphery after tumor cell inoculation. Single cell analysis of immature neutrophils cross human tumor, bone marrow and HIS mouse bone marrow samples revealed FCN1 as a common marker for this heterogeneous population. Finally, we proved that elimination of bone marrow immature neutrophils with Flt3L treatment reduced tumor associated neutrophils and delays tumor growth in NCG-Gfi1 −/−HIS mice. In summary, human immunosuppressive and tumor-promoting neutrophils are predominantly immature neutrophils and targeting this population in bone marrow may represent a novel strategy for cancer immunotherapy This study was supported by grants from the National Key Research and Development Program of China (2019YFA0802900), and National Natural Science Foundation of China (32070942, 32122035, 32000669).

Unraveling novel crosstalk between distinct neutrophil subsets and soluble plasma factors in sepsis and ARDS

Abstract Sepsis is a life-threatening condition caused by an aberrant immune response to infection and subsequent organ dysfunction. Further, due to shared mechanisms such as severe inflammation and endothelial dysfunction, sepsis remains the most common etiology in acute respiratory distress syndrome (ARDS) patients. Despite multiple efforts to identify a distinct signature to support the development of new therapeutics, sepsis remains a significant unmet clinical need. While singular components of the dysregulated immune response have been explored, studies linking different data layers are lacking. Using an integrated systems immunology approach, we evaluated neutrophil phenotypes and related changes in soluble plasma factors in sepsis versus healthy donors. Results from our studies identify differentially expressed mature and immature neutrophil subsets in sepsis patients. Further, we show that expression of neutrophil CD10 is associated with factors such as pentraxin-3, angiopoietin-2, and lysophosphatidylcholines, in the sepsis cohort. These findings detail a novel correlation between increased presence of immature neutrophils and endothelial dysfunction/metabolic alterations in sepsis patients. Additionally, we profiled the neutrophil landscape and evaluated levels of soluble plasma factors, in a subset of ARDS patients and healthy donors. Here, we present distinct findings that reveal a novel crosstalk between neutrophil subsets and parameters indicative of endothelial dysfunction, in sepsis and ARDS patients. Overall, our study reinforces the importance of data integration and provides novel insights that could help inform future mechanistic studies, early patient stratification and treatment options.

SEASONAL VARIABILITY OF IMMUNOHEMATOLOGICAL PARAMETERS OF PERPHERAL BLOOD OF THE BAT <i>MYOTIS DASYCNEME</i> (BOIE, 1825) LIVING IN THE URALS

A comparative analysis of the immunohematological parameters of bats from fauna of the Urals Myotis dasycneme (Boie, 1825) in seasonal periods of their annual life cycle is presented for the first time. Multivariate nonparametric analysis of variance showed the absence of significant gender differences in bats according to the studied parameters of red blood (p = 0.35). The sexual determination of bats was established by the content of leukocytes, band neutrophils and segments neutrophils, lymphocytes in the blood (p 0.05). Males are characterized by a higher development of innate immunity in the summer compared to females (p 0.05). In the autumn-winter hibernation period, males and females have a significant lymphocytic profile (50.6–53.5%), providing immune “supervision” and specifically reactive activation of the acquired adaptive immune response.

Microenvironmental control of hematopoietic stem cell fate via CXCL8 and protein kinase C

Altered hematopoietic stem cell (HSC) fate underlies primary blood disorders but microenvironmental factors controlling this are poorly understood. Genetically barcoded genome editing of synthetic target arrays for lineage tracing (GESTALT) zebrafish were used to screen for factors expressed by the sinusoidal vascular niche that alter the phylogenetic distribution of the HSC pool under native conditions. Dysregulated expression of protein kinase C delta (PKC-δ, encoded by prkcda) increases the number of HSC clones by up to 80% and expands polyclonal populations of immature neutrophil and erythroid precursors. PKC agonists such as cxcl8 augment HSC competition for residency within the niche and expand defined niche populations. CXCL8 induces association of PKC-δ with the focal adhesion complex, activating extracellular signal-regulated kinase (ERK) signaling and expression of niche factors in human endothelial cells. Our findings demonstrate the existence of reserve capacity within the niche that is controlled by CXCL8 and PKC and has significant impact on HSC phylogenetic and phenotypic fate.

ALKBH5-mediated m 6A RNA demethylation promotes antibacterial innate defense by intrinsically driving neutrophil generation and recruitment

Abstract Neutrophil generation and effective recruitment into the site of infection are essential for innate immune defense against bacterial infection. These processes that directed by extracellular signals such as growth factors or chemokines have been extensively studied, yet the intrinsic and new mechanism for determining neutrophil generation and recruitment needs further investigation. N6-methyladenosine (m 6A) RNA modification and its demethylase alkB homolog 5 (ALKBH5) are key epigenetic regulators of immunity and inflammation, whereas their role in neutrophil generation and recruitment remains unclear. Here, using cecal ligation and puncture-induced sepsis to model systemic bacterial infection, we found that ALKBH5 is required for antibacterial innate defense by intrinsically inducing neutrophil generation and recruitment. Indeed, Alkbh5-deficient mice exhibited increased mortality, higher bacterial burden and proinflammatory cytokine production than wild-type littermates undergoing sepsis. Meanwhile, ALKBH5 depletion significantly impaired the generation of immature neutrophils in bone marrow and also reduced neutrophil recruitment into the infected site of sepsis mice. Mechanistically, ALKBH5 imprinted generation- and recruitment-promoting transcriptome signatures in both mouse and human neutrophils during bacterial infection, specially modulated the protein expression of SOCS3 and CXCR2 through altering m 6A methylation-mediated decay of their mRNAs. Together, our findings reveal a previously unknown role of ALKBH5 in intrinsically driving neutrophil generation and recruitment, indicating neutrophil m 6A RNA modification as potential target for controlling bacterial infections. Supported by grants from the National Natural Science Foundation of China (81788101, 82071793), the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2021-I2M-1-017), the Beijing Natural Science Foundation (7212069), and the Beijing Nova Program (20220484065).

Serum and erythrocyte oxidative stress in dogs with acanthocytosis

Oxidative stress occurs as a result of an increase in oxidants and/or a reduction of antioxidants in the body, causing oxidation of cellular constituents and cellular dysfunction, which can lead to premature cell death. This study aimed to evaluate serum and red blood cell (RBC) oxidative stress in dogs with acanthocytosis. Sixteen dogs with acanthocytosis confirmed on blood smear, regardless of the associated pathological condition, and 17 healthy dogs without clinical and laboratorial alterations were selected. Complete blood counts (CBC) were performed by an automated cell counter, biochemical and oxidative stress analyzes were performed by an automated photocolorimeter. Dogs with acanthocytosis showed lower RBC, hemoglobin, hematocrit and mean corpuscular hemoglobin concentration (MCHC), and higher red cell distribution width (RDW) compared with healthy dogs. On the leukogram, higher concentrations of band neutrophils and lower numbers of lymphocytes were observed in dogs with acanthocytosis. On serum biochemical parameters, a significant increase in ALP activity, cholesterol and globulin levels, and decreased albumin concentration were observed in dogs with acanthocytosis. Regarding oxidative stress parameters, dogs with acanthocytosis showed lower serum total antioxidant capacity (TAC) by cupric reduction (TAC-CUPRAC) and higher TAC by ferric reduction (TAC-FRAP), total oxidant capacity (TOC) and lipid peroxidation, while RBC showed higher TAC by ABTS cation reduction alone (TAC-ABTS) and associated with peroxidase (TAC-ABTS+HRP), TAC-CUPRAC, TOC and lipid peroxidation compared with those of control dogs. Dogs with acanthocytosis demonstrate serum and erythrocyte oxidative stress, which may be one of the factors involved in erythrocyte damage.

Neutrophils and emergency granulopoiesis drive immune suppression and an extreme response endotype during sepsis.

Sepsis arises from diverse and incompletely understood dysregulated host response processes following infection that leads to life-threatening organ dysfunction. Here we showed that neutrophils and emergency granulopoiesis drove a maladaptive response during sepsis. We generated a whole-blood single-cell multiomic atlas (272,993 cells, n = 39 individuals) of the sepsis immune response that identified populations of immunosuppressive mature and immature neutrophils. In co-culture, CD66b+ sepsis neutrophils inhibited proliferation and activation of CD4+ T cells. Single-cell multiomic mapping of circulating hematopoietic stem and progenitor cells (HSPCs) (29,366 cells, n = 27) indicated altered granulopoiesis in patients with sepsis. These features were enriched in a patient subset with poor outcome and a specific sepsis response signature that displayed higher frequencies of IL1R2+ immature neutrophils, epigenetic and transcriptomic signatures of emergency granulopoiesis in HSPCs and STAT3-mediated gene regulation across different infectious etiologies and syndromes. Our findings offer potential therapeutic targets and opportunities for stratified medicine in severe infection.

Identification of a sub-group of critically ill patients with high risk of intensive care unit-acquired infections and poor clinical course using a transcriptomic score

BackgroundThe development of stratification tools based on the assessment of circulating mRNA of genes involved in the immune response is constrained by the heterogeneity of septic patients. The aim of this study is to develop a transcriptomic score based on a pragmatic combination of immune-related genes detected with a prototype multiplex PCR tool.MethodsAs training cohort, we used the gene expression dataset obtained from 176 critically ill patients enrolled in the REALISM study (NCT02638779) with various etiologies and still hospitalized in intensive care unit (ICU) at day 5–7. Based on the performances of each gene taken independently to identify patients developing ICU-acquired infections (ICU-AI) after day 5–7, we built an unweighted score assuming the independence of each gene. We then determined the performances of this score to identify a subgroup of patients at high risk to develop ICU-AI, and both longer ICU length of stay and mortality of this high-risk group were assessed. Finally, we validated the effectiveness of this score in a retrospective cohort of 257 septic patients.ResultsThis transcriptomic score (TScore) enabled the identification of a high-risk group of patients (49%) with an increased rate of ICU-AI when compared to the low-risk group (49% vs. 4%, respectively), with longer ICU length of stay (13 days [95% CI 8–30] vs. 7 days [95% CI 6–9], p < 0.001) and higher ICU mortality (15% vs. 2%). High-risk patients exhibited biological features of immune suppression with low monocytic HLA-DR levels, higher immature neutrophils rates and higher IL10 concentrations. Using the TScore, we identified 160 high-risk patients (62%) in the validation cohort, with 30% of ICU-AI (vs. 18% in the low-risk group, p = 0.06), and significantly higher mortality and longer ICU length of stay.ConclusionsThe transcriptomic score provides a useful and reliable companion diagnostic tool to further develop immune modulating drugs in sepsis in the context of personalized medicine.

Complement dependent TNFα production in neutrophil-like HL60 cells

Neutrophils develop in the bone marrow (BM) from hematopoietic stem cells (HSCs) through a series of progenitor cells and mature neutrophils play a critical role in the human immune system. Previous studies revealed that tumor necrosis factor α (TNFα) produced by immature neutrophils contributes to HSCs development and vascular regeneration in the BM niche. However, the precise mechanism of TNFα production in immature neutrophils remains unclear. This study aims to assess the relationship between complement C3 activation and TNFα production from immature neutrophils. We investigated the regulatory mechanism of TNFα production by complement components in neutrophil-like HL60 cells. Flow cytometric analysis showed that C3a receptor (C3aR) and C3bi receptor (CR3, Mac-1, CD11b/CD18, integrin αMβ2) are expressed on the surface of neutrophil-like HL60 cells. We found that zymosan-treated human serum leads to TNFα production in neutrophil-like HL60 cells, but not in human polymorphonuclear cells (PMNs). A C3-convertase inhibitor, compstatin suppresses TNFα production. These data suggest that the TNFα production is mediated by complement C3 activation. Furthermore, the TNFα production is enhanced by Ca2+ elevating agents, thapsigargin (TG), but is suppressed by treatment with Ca2+ chelators, EGTA, or BAPTA-AM. In addition, the soluble TNFα production is suppressed by treatment with immobilized-fibrinogen or -fibronectin. Thus, the TNFα production is enhanced by intracellular Ca2+ elevation and is negatively regulated by the interaction between the neutrophil-like HL60 cells and fibrinogen or fibronectin.

Regulation of granulocyte colony-stimulating factor-induced hematopoietic stem cell mobilization by the sympathetic nervous system.

Granulocyte colony-stimulating factor (G-CSF) is now a standard agent to mobilize hematopoietic stem cells (HSCs) from the bone marrow to circulation. This review introduced mechanistic insights from the aspect of the sympathetic nervous system (SNS). Mobilization efficiency is determined by the balance between promotion and suppression pathways critically regulated by the SNS. G-CSF-induced high catecholaminergic tone promotes mobilization by (1) the strong suppression of osteolineage cells as a hematopoietic microenvironment and (2) fibroblast growth factor 23 production from erythroblasts, which inhibits CXCR4 function in HSCs. Simultaneously, SNS signals inhibit mobilization by (1) prostaglandin E2 production from mature neutrophils to induce osteopontin in osteoblasts to anchor HSCs and (2) angiopoietin-like protein 4 production from immature neutrophils via peroxisome proliferator-activated receptor δ to inhibit BM vascular permeability. We now know not only the regulatory mechanisms of G-CSF-induced mobilization but also the leads about unfavorable clinical phenomena, such as low-grade fever, bone pain, and poor mobilizers. Recent understanding of the mechanism will assist clinicians in the treatment for mobilization and researchers in the studies of the hidden potential of BM.

Re-estimation of juvenile's Isurus oxyrinchus growth in the Mexican Pacific through a multi-model inference approach and verification of growth band periodicity.

An update of the age and growth for juveniles of shortfin mako shark (Isurus oxyrinchus) from the Mexican Pacific is presented, based on the analysis of growth band counts from dorsal vertebrae of 198 individuals (110 females 74-231 cm of total length, TL, and 88 males 72-231 cm TL) caught during 2008-2018. New available information on vertebral growth band periodicity (biannual deposition in juveniles) and the convenience of using vertebrae form the dorsal region over cervical region to count growth bands, as well as a multi-model approach, were used. The von Bertalanffy growth model (VB), Gompertz, logistic and the two parameters of VB were fitted to the length-at-age. Only the ages ≤ 6 years were used for the fitting of the models and their performance was compared with the small-sample bias-corrected form of Akaike information criterion (AICc), their differences and weights . Following a multi-model inference approach, the model averaged asymptotic length , length-at-age 0 and their unconditional standard error , were estimated for each sex scenario using the 3-parameters version of each model.The precision of growth band counts was acceptable for the different methods used and by two different readers. The Centrum edge analysis (CEA) and marginal increment analysis (MIA) did not support the hypothesis of biannual band pair formation for juveniles, likewise for adults the periodicity could not be verified due to the low sample of large animals. Still, age was estimated assuming the formation of two pairs of growth bands per year during the first five years of age and one pair of bands per year afterwards considering direct validation information. The estimated ages ranged from 0-14 for females and 0-6 for males. The Kimura's likelihood ratio test showed no differences in the growth curves of juveniles by sex (P>0.05). According to the AICc, the 2-VB better fitted the length-at-age data for sex combined (Linf = 386.4 cm, k = 0.12 yr-1 , L0 = 70 cm). The model averaged and were 378.3 cm ( = 64.5) and 69.5 cm = 6.3) respectively. The growth parameters determined for juveniles of I. oxyrinchus are similar with those estimated in other regions, showing relatively fast growth rate as previously reported, medium longevity in comparison to other shark species and natural mortality close to those reported in the last stock assessment for the North Pacific Ocean. These life-history parameters should be considered to evaluate the population in the region to develop better fishery management and conservation measures.

Abstract 2877: Profiling of myeloid-derived suppressor cells in Pten-deficient in prostate cancer progression

Abstract Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of immature immune cells that expand during cancer and can induce potent immune suppression. These cells play critical roles in supporting tumor growth and progression and their induction has been associated with the inactivation of PTEN. To date, few therapies targeting these cells are available which is partly due to their increasingly complex nature and context-dependent functions. Moreover, there is no clear definition to identify these cells. Enhancing our understanding of this cell population will aid in developing more effective cancer therapies. Here, we used gene expression profiling, flow cytometry, and quantitative immunohistochemistry to characterize tumor-infiltrating and circulating MDSCs in prostate carcinogenesis and progression in transgenic mouse models of prostate cancer driven by the conditional inactivation of Pten and Trp53. Transcriptomic profiling revealed enrichment of various myeloid-derived cell signatures in the early stages of prostate cancer including MDSCs, macrophages, and neutrophils. Flow cytometric analysis confirmed the increased infiltration of CD11b+/Ly6G+ (polymorphonuclear MDSCs (pMDSCs)) and CD11b+/Ly6C+ (monocytic MDSCs (mMDSCs)) cells in prostate tumors. The temporal dynamics of tumor-infiltrating myeloid cells were further profiled and showed that pMDSC and mMDSC abundances were 1.6 and 5.7-fold higher, respectively, in older mice with late-stage locally invasive adenocarcinomas (AdCa) compared to younger mice with early-stage prostatic intraepithelial neoplasia (PIN). In both instances, pMDSCs comprised most of the leukocyte infiltrate, however, mMDSCs tended to be positively correlated with tumor burden in AdCa but not PIN. In peripheral blood, CD44+/CD62L+ immature neutrophils were associated with high tumor burden in aged mice. Treatments targeting the androgen receptor (AR) signaling further exacerbated pMDSC infiltration in tumors and was associated with resistance to second androgen receptor-targeted agent (ARTA) therapy in a model of castration resistant prostate cancer. Our findings indicate that MDSCs are a prominent population in Pten-null prostate cancer and are implicated with resistance to prostate cancer treatment. MDSCs constitute a potentially targetable population and these models serve as viable platforms to further investigate their biological activity and further evaluate novel MDSC-directed therapies. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sako, Naomi Ando, Kazuko Sakai, Eri Banno, Shogo Adomi, Mitsuhisa Nishimoto, Takafumi Minami, Yasunori Mori, Kazutoshi Fujita, Masahiro Nozawa Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. Profiling of myeloid-derived suppressor cells in Pten-deficient in prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2877.

Abstract 5654: A therapeutic humanized anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human granulocytic myeloid-derived suppressor cells (gMDSCs) and regulatory T (Tregs) cells

Abstract Background: NEO-201 is a humanized IgG1 mAb reactive against multiple human cancers but not against most normal epithelial tissues. NEO-201 binds to core 1 or extended core 1 O-glycans expressed by its target cells, including neutrophils, various carcinomas, and some human hematological malignancies. NEO-201 can mediate antitumor activity through antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and blockade of the CEACAM5/CEACAM1 ICI pathway. A previous study using flow cytometry demonstrated that NEO-201+/CD4+ T cells were also CD25+/CD127−/Foxp3+/CD15s+ using PBMCs from healthy donors (HD). NEO-201 can kill these Treg cells through CDC in vitro. NEO-201 does not bind to the majority of CD4+ T cells and to other immune subsets. Human gMDSCs are increased in cancer patients and are a population of immature MDSCs deriving from immature neutrophils and alternative activation of mature neutrophils. gMDSC are characterized by HLA-DR−, CD11b+, CD33+, CD15+phenotype.We have shown that NEO-201 recognizes and kill human neutrophils through ADCC. This current investigation was designed to evaluate whether NEO-201 can target and mediate ADCC activity against human gMDSCs. Methods: gMDSCs were generated from human neutrophils from 5 HD isolated using EasySepTM direct human neutrophil isolation kit. Isolated neutrophils were cultured in complete RPMI1640 medium supplemented with human GM-CSF and human IL-6 for 7 days. Phenotypic analysis by flow cytometry was performed on the generated gMDSCs using NEO-201 and mAbs against human CD33, HLA-DR, CD15, CD14, CD66b. Flow cytometry based ADCC assay was performed using gMDSCs stained with both CD33 and HLA-DR as target. PBMCs from a separate HD were used as effectors at different E:T ratios. The ADCC activity of NEO-201 was evaluated comparing the percentage of CD33+/HLA-DR− viable cells in gMDSCs incubated with medium alone to the percentage of CD33+/HLA-DR− viable cells incubated with PBMCs alone and with PBMCs plus NEO-201. Results: Flow cytometry analysis revealed that gMDSCs can be generated from human neutrophils after 7 days of culture with GM-CSF and IL-6 and that they express the following phenotype: HLA-DR−/CD33+/CD15+/CD14−/CD66b+. NEO-201 bound to the majority of these gMDSCs. NEO-201 was functional in mediating ADCC to kill these gMDSCs. Conclusion: This study demonstrated that NEO-201 can be used to identify and kill suppressive gMDSCs in addition to Treg cells. Depletion of suppressive Tregs and gMDSCs in the TME could be an effective strategy to prevent hyperprogressive disease when anti-PD-1 is used in cancer immunotherapy. These data support the rationale for the ongoing phase II clinical trial using NEO-201 in combination with pembrolizumab in checkpoint refractory patients with metastatic solid tumors. Citation Format: Massimo Fantini, Christina M. Annunziata, Philip M. Arlen, Kwong Y. Tsang. A therapeutic humanized anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human granulocytic myeloid-derived suppressor cells (gMDSCs) and regulatory T (Tregs) cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5654.

Abstract 5506: Circulating immature neutrophils early detect hyperprogressive disease upon first-line PD-1/PD-L1 inhibitors in non-small cell lung cancer patients selecting best candidates for platinum-based chemotherapy and PD-1/PD-L1 inhibitors combinations

Abstract Background: Hyperprogressive disease (HPD) has been described in ≃14-25% of pretreated non-small cell lung cancer (NSCLC) patients upon single-agent (SA) PD-1/PD-L1 inhibitors (ICI) and has not been reported upon platinum-based chemotherapy (PCT) and ICI combinations. So far, no predictive biomarkers are available for HPD early detection. Methods: NSCLC patients treated with 1st line SA-ICI or PCT-ICI were assessed for HPD and circulating neutrophils. HPD was defined as delta tumor growth rate (TGR) &amp;gt;50% and/or TGR ratio ≥2. Circulating low density neutrophils (LDNs) were assessed by flow cytometry on peripheral blood mononuclear cells (PMBCs). LDNs were defined as CD66b+CD15+ cells among CD11b+ PBMCs and immature subtypes as CD10− LDNs. The LDNs predictive role was assessed by penalized model-based tests. Results: 144 NSCLC patients were included: 75 treated with SA-ICI, 69 with PCT-ICI. In the SA-ICI cohort, HPD occurred in 8 (11%) patients, while progressive disease (PD) and response or stable disease (PR/SD) occurred in 33 (44%) and 34 (45%) of patients respectively. Immature circulating CD10− LDNs were significantly higher in baseline blood samples of HPD patients [median (ME): 39.3, interquartile range (IQR): 28.7] compared to PD [ME: 7.4, IQR: 14.9, p&amp;lt;0.01] or PR/SD patients [ME: 3.7, IQR: 12.6, p&amp;lt;0.01]. Circulating CD10− LDNs were associated with HPD [odds ratio (OR): 1.17, 95% CI: 1.06; 1.29], with a good prediction capability [cross-validated AUC 0.97 (95%CI: 0.94;1.00)]. A 30.5% cut-off value for CD10− LDNs circulating neutrophils was identified by Younden index to discriminate HPD from others. In the PCT-ICI cohort, 14 patients had circulating CD10− LDNs ≥30.5%, being at high risk of HPD. However, no HPD was observed in PCT-ICI cohort and dynamic LDNs evaluation in high HPD risk patients showed 52.3% (IQR: 28.4) median reduction in CD10− LDNs upon PCT-ICI, versus only 8.9% (IQR: 34.6) reduction in HPD patients upon SA-ICI, suggesting that PCT prevents HPD by reducing selectively immature LDNs. Conclusions: Baseline circulating immature neutrophils characterize HPD upon 1st line SA-ICI and a 30.5% cut-off of immature neutrophils could select NSCLC patients to be addressed to PCT-ICI combinations. Citation Format: Roberto Ferrara, Giuseppe Lo Russo, Chiara Maura Ciniselli, Annamaria Piva, Barbara Bassani, Elena Jachetti, Giuseppina Calareso, Valeria Duroni, Settimio Di Gregorio, Claudia Proto, Arsela Prelaj, Alessandro De Toma, Mario Occhipinti, Marta Brambilla, Sara Manglaviti, Laura Mazzeo, Arturo Rinaldi, Teresa Beninato, Monica Ganzinelli, Filippo De Braud, Marina Chiara Garassino, Paolo Verderio, Mario Paolo Colombo, Sabina Sangaletti. Circulating immature neutrophils early detect hyperprogressive disease upon first-line PD-1/PD-L1 inhibitors in non-small cell lung cancer patients selecting best candidates for platinum-based chemotherapy and PD-1/PD-L1 inhibitors combinations. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5506.

CD11c regulates neutrophil maturation.

Sepsis continues to be associated with high morbidity and mortality. Currently, sepsis is managed only conservatively. In sepsis, a substantial number of neutrophils is required, leading to accelerated neutrophil production. Immature neutrophils are released into the circulation to meet a demand, despite their less effective functioning in microbial eradication. Although an intervention to provide more mature neutrophils may serve as a potential sepsis treatment, the mechanism of neutrophil differentiation and maturation remains poorly understood. We discovered that CD11c, traditionally known as a dendritic cell marker, was expressed in neutrophils and regulated neutrophil maturation and effector functions. In the absence of CD11c, neutrophil maturation was impaired in the bone marrow, concomitant with a significant increase in the proliferation and apoptosis of preneutrophils, associated with less effector functions. Under lipopolysaccharide challenge, inducing an emergent neutrophil production in the bone marrow, CD11c deficiency exaggerated the release of immature neutrophils into the circulation, associated with a significant proliferation and apoptosis of preneutrophils. In contrast, constitutively active CD11c knock-in mice showed accelerated neutrophil maturation associated with enhanced effector functions, which further supports the notion that CD11c regulates neutrophil maturation. Furthermore, the constitutively active CD11c knock-in mice offered enhanced bacterial eradication. Taken together, we discovered that CD11c was critical for the regulation of neutrophil maturation, and CD11c activation could serve as a potential target for sepsis treatment.

Development of a Nomogram to Estimate the 60-Day Probability of Death or Culling Due to Severe Clinical Mastitis in Dairy Cows at First Veterinary Clinical Evaluation

Severe clinical mastitis is a frequent disease of dairy cattle. An effective mean of predicting survival despite treatment would be helpful for making euthanasia decisions in poor prognosis cases. The objective was to develop a nomogram for prediction of death or culling in the 60 days following a severe mastitis episode in dairy cows at first veterinary visit in farm settings. A total of 224 dairy cows presenting severe clinical mastitis and examined for the first time by a veterinarian were included in a prospective study. Clinical and laboratory (complete blood cell count, L-lactate, cardiac troponin I, milk culture) variables were recorded. Animals were followed for 60 days. A nomogram was built with an adaptive elastic-net Cox proportional hazards model. Performances and relevance were evaluated by area under the receiver operating characteristic curve (AUC), Harrell's concordance index (C-index), calibration curve, decision curve analysis (DCA) and misclassification cost term (MCT). The nomogram included: lactation number, recumbency, depression intensity, capillary refilling time, ruminal motility rate, dehydration level, lactates concentration, hematocrit, band neutrophils count, monocyte count, and milk bacteriology. The AUC and C-index showed a good calibration and ability to discriminate. The DCA suggested that the nomogram was clinically relevant. Euthanizing animals having less than 25% probability of survival is economically optimal. It could be used for early euthanasia decisions in animals that would not survive despite treatment. To facilitate the use of this nomogram by veterinarians, a web-based app was developed.

Assessment of neutrophil subsets and immune checkpoint inhibitor expressions on T lymphocytes in liver transplantation: A preliminary study beyond the neutrophil-lymphocyte ratio.

Background: Advanced stages of cirrhosis are characterized by the occurrence of progressive immune alterations known as CAID (Cirrhosis Associated Immune Dysfunction). In advanced cirrhosis, liver transplantation (LT) remains the only curative treatment. Sepsis, shares many similarities with decompensated cirrhosis in terms of immuno-inflammatory response. In both conditions, the neutrophil-lymphocyte ratio (NLR) is associated with poor outcomes. Based on alterations in sepsis, we hypothesized that we could observe in cirrhotic and LT patients more detailed neutrophil and lymphocyte phenotypes. To this end, along with leukocyte count, we assessed immature neutrophils, LOX-1+ MDSC and PD-1 and TIM-3 lymphocyte expressions in cirrhotic patients before transplantation in association with liver disease severity and during the first month after transplantation. Methods: We conducted a prospective monocentric study including cirrhotic patients registered on LT waiting-list. Blood samples were collected at enrolment before LT and for 1month post-LT. In addition to NLR, we assessed by whole blood flow cytometry the absolute count of immature neutrophils and LOX-1+ MDSC as well as the expressions of immune checkpoint receptors PD-1 and TIM-3 on T lymphocytes. Results: We included 15 healthy volunteers (HV) and 28 patients. LT was performed for 13 patients. Pre-LT patients presented with a higher NLR compared to HV and NLR was associated with cirrhosis severity. Increased immature neutrophils and LOX-1+ MDSC counts were observed in the most severe patients. These alterations were mainly associated with acute decompensation of cirrhosis. PD-1 and TIM-3 expressions on T lymphocytes were not different between patients and HV. Post-LT immune alterations were dominated by a transitory but tremendous increase of NLR and immature neutrophils during the first days post-LT. Then, immune checkpoint receptors and LOX-1+ MDSC tended to be overexpressed by the second week after surgery. Conclusion: The present study showed that NLR, immature neutrophils and LOX-1+ MDSC counts along with T lymphocyte count and checkpoint inhibitor expression were altered in cirrhotic patients before and after LT. These data illustrate the potential interest of immune monitoring of cirrhotic patients in the context of LT in order to better define risk of sepsis. For this purpose, larger cohorts of patients are now necessary in order to move forward a more personalised care of LT patients.

Dynamic changes in radiological parameters, immune cells, selected miRNAs, and cytokine levels in peripheral blood of patients with severe COVID‑19.

The present study aimed to investigate the dynamic changes in peripheral blood leucocyte subpopulations, cytokine and miRNA levels, and changes in computed tomography (CT) scores in patients with severe coronavirus disease 2019 (COVID-19) (n=14) and age-matched non-COVID-19 volunteers (n=17), which were included as a reference control group. All data were collected on the day of patient admission (day 0) and on the 7th, 14th and 28th days of follow-up while CT of the lungs was performed on weeks 2, 8, 24 and 48. On day 0, lymphopenia and leucopenia were detected in most patients with COVID-19, as well as an increase in the percentage of banded neutrophils, B cells, and CD4+ Treg cells, and a decrease in the content of PD-1low T cells, classical, plasmacytoid, and regulatory dendritic cells. On day 7, the percentage of T and natural killer cells decreased with a concurrent increase in B cells, but returned to the initial level after treatment discharge. The content of different T and dendritic cell subsets among CD45+ cells increased during two weeks and remained elevated, suggesting the activation of an adaptive immune response. The increase of PD-1-positive subpopulations of T and non-T cells and regulatory CD4 T cells in patients with COVID-19 during the observation period suggests the development of an inflammation control mechanism. The levels of interferon γ-induced protein 10 (IP-10), tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 decreased on day 7, but increased again on days 14 and 28. C-reactive protein and granulocyte colony-stimulating factor (G-CSF) levels decreased gradually throughout the observation period. The relative expression levels of microRNA (miR)-21-5p, miR-221-3p, miR-27a-3p, miR-146a-5p, miR-133a-3p, and miR-126-3p were significantly higher at the beginning of hospitalization compared to non-COVID-19 volunteers. The plasma levels of all miRs, except for miR-126-3p, normalized within one week of treatment. At week 48, CT scores were most prominently correlated with the content of lymphocytes, senescent memory T cells, CD127+ T cells and CD57+ T cells, and increased concentrations of G-CSF, IP-10, and macrophage inflammatory protein-1α.

Dysbiosis of a microbiota–immune metasystem in critical illness is associated with nosocomial infections

Critically ill patients in intensive care units experience profound alterations of their gut microbiota that have been linked to a high risk of hospital-acquired (nosocomial) infections and adverse outcomes through unclear mechanisms. Abundant mouse and limited human data suggest that the gut microbiota can contribute to maintenance of systemic immune homeostasis, and that intestinal dysbiosis may lead to defects in immune defense against infections. Here we use integrated systems-level analyses of fecal microbiota dynamics in rectal swabs and single-cell profiling of systemic immune and inflammatory responses in a prospective longitudinal cohort study of critically ill patients to show that the gut microbiota and systemic immunity function as an integrated metasystem, where intestinal dysbiosis is coupled to impaired host defense and increased frequency of nosocomial infections. Longitudinal microbiota analysis by 16s rRNA gene sequencing of rectal swabs and single-cell profiling of blood using mass cytometry revealed that microbiota and immune dynamics during acute critical illness were highly interconnected and dominated by Enterobacteriaceae enrichment, dysregulated myeloid cell responses and amplified systemic inflammation, with a lesser impact on adaptive mechanisms of host defense. Intestinal Enterobacteriaceae enrichment was coupled with impaired innate antimicrobial effector responses, including hypofunctional and immature neutrophils and was associated with an increased risk of infections by various bacterial and fungal pathogens. Collectively, our findings suggest that dysbiosis of an interconnected metasystem between the gut microbiota and systemic immune response may drive impaired host defense and susceptibility to nosocomial infections in critical illness.