Classical Monocytes Research Articles

INFLAMMAGING FROM THE PERSPECTIVE OF GYNECOLOGICAL ENDOCRINOLOGY. STARTING POINT

BACKGROUND: It is known that the aging of the immune system in individuals of both sexes is a natural process of ontogenesis, and in women it is apparently identified with the entry into postmenopause and a decrease in ovarian function. AIM: To study immune blood parameters in peri- and early postmenopausal women. METHODS: The single-center, cross-sectional study included 50 women aged 45 to 59 years in the phase of reproductive aging, peri- and early postmenopause. Major subpopulations of blood cells such as cytotoxic T lymphocytes, T helper cells, NK cells, B lymphocytes, classical, non-classical and intermediate monocytes, as well as pro- and anti-inflammatory markers on the isolated monocyte populations were analyzed using flow cytometry. A multiplex assay was used to detect 27 plasma cytokines. RESULTS: Reproductive aging in women during the transition stage of reproductive aging from peri- to postmenopause is accompanied by an increase in the monocyte-associated inflammatory reaction and humoral response, which is expressed in the redistribution of the monocyte population towards non-classical monocytes (p = 0.034) and an increase in the level of B-lymphocytes by 1.8 times (p=0.023), as well as a significant (p=0.022) increase in the level of MCP-1, a marker associated with inflammation. CONCLUSION: Considering the pro-inflammatory nature of changes associated with immune aging in women, it seems preferable to start “early” menopausal hormone therapy (MHT) during perimenopause.

Single-cell landscape revealed immune characteristics associated with disease phases in brucellosis patients.

A comprehensive immune landscape for Brucella infection is crucial for developing new treatments for brucellosis. Here, we utilized single-cell RNA sequencing (scRNA-seq) of 290,369 cells from 35 individuals, including 29 brucellosis patients from acute (n = 10), sub-acute (n = 9), and chronic (n = 10) phases as well as six healthy donors. Enzyme-linked immunosorbent assays were applied for validation within this cohort. Brucella infection caused a significant change in the composition of peripheral immune cells and inflammation was a key feature of brucellosis. Acute patients are characterized by potential cytokine storms resulting from systemic upregulation of S100A8/A9, primarily due to classical monocytes. Cytokine storm may be mediated by activating S100A8/A9-TLR4-MyD88 signaling pathway. Moreover, monocytic myeloid-derived suppressor cells were the probable contributors to immune paralysis in acute patients. Chronic patients are characterized by a dysregulated Th1 response, marked by reduced expression of IFN-γ and Th1 signatures as well as a high exhausted state. Additionally, Brucella infection can suppress apoptosis in myeloid cells (e.g., mDCs, classical monocytes), inhibit antigen presentation in professional antigen-presenting cells (APCs; e.g., mDC) and nonprofessional APCs (e.g., monocytes), and induce exhaustion in CD8+ T/NK cells, potentially resulting in the establishment of chronic infection. Overall, our study systemically deciphered the coordinated immune responses of Brucella at different phases of the infection, which facilitated a full understanding of the immunopathogenesis of brucellosis and may aid the development of new effective therapeutic strategies, especially for those with chronic infection.

Elucidating the Immune Response to SARS-CoV-2: Natural Infection versus Covaxin/Covishield Vaccination in a South Indian Population.

A natural infection or a vaccination can initially prime the immune system to form immunological memory. The immunity engendered by vaccination against COVID-19 versus natural infection with SARS-CoV-2 has not been well studied in the Indian population. In this study, we compared the immunity conferred by COVID-19 vaccines to naturally acquired immunity to SARS-CoV-2 in a South Indian population. We examined binding and neutralizing antibody (NAb) levels against the ancestral and variant lineages and assessed the ex vivo cellular parameters of memory T cells, memory B cells, and monocytes and finally measured the circulating cytokine response. COVID-19 vaccination stimulates heightened levels of IgG antibodies against the original strain of SARS-CoV-2, as well as increased binding to the spike protein and neutralizing antibody levels. This enhanced response extends to variant lineages such as B.1.617.2 (Delta, India), B.1.1.529 (Omicron, India), B.1.351 (Beta, South Africa), and B.1.1.7 (Alpha, UK). COVID-19 vaccination differs from SARS-CoV-2 infection by having increased frequencies of classical memory B cells, activated memory B and plasma cells, CD4/CD8 T cells of effector memory, effector cells, stem cell-like memory T cells, and classical and intermediate monocytes and diminished frequencies of CD4/CD8 T cells of central memory and non-classical monocytes in vaccinated individuals in comparison to those with natural infection. Thus, COVID-19 vaccination is characterized by enhanced humoral responses and robust activation of innate and memory T cell responses in comparison to natural infection in a South Indian population.

A subpopulation of human bone marrow erythroid cells displays a myeloid gene expression signature similar to that of classic monocytes.

Erythroid cells, serving as progenitors and precursors to erythrocytes responsible for oxygen transport, were shown to exhibit an immunosuppressive and immunoregulatory phenotype. Previous investigations from our research group have revealed an antimicrobial gene expression profile within murine bone marrow erythroid cells which suggested a role for erythroid cells in innate immunity. In the present study, we focused on elucidating the characteristics of human bone marrow erythroid cells through comprehensive analyses, including NanoString gene expression profiling utilizing the Immune Response V2 panel, a BioPlex examination of chemokine and TGF-beta family proteins secretion, and analysis of publicly available single-cell RNA-seq data. Our findings demonstrate that an erythroid cell subpopulation manifests a myeloid-like gene expression signature comprised of antibacterial immunity and neutrophil chemotaxis genes which suggests an involvement of human erythroid cells in the innate immunity. Furthermore, we found that human erythroid cells secreted CCL22, CCL24, CXCL5, CXCL8, and MIF chemokines. The ability of human erythroid cells to express these chemokines might facilitate the restriction of immune cells in the bone marrow under normal conditions or contribute to the ability of erythroid cells to induce local immunosuppression by recruiting immune cells in their immediate vicinity in case of extramedullary hematopoiesis.

Beta2 adrenergic receptor-mediated abnormal myelopoiesis drives neuroinflammation in aged patients with traumatic brain injury.

Aged patients often suffer poorer neurological recovery than younger patients after traumatic brain injury (TBI), but the mechanisms underlying this difference remain unclear. Here, we demonstrate abnormal myelopoiesis characterized by increased neutrophil and classical monocyte output but impaired nonclassical patrolling monocyte population in aged patients with TBI as well as in an aged murine TBI model. Retrograde and anterograde nerve tracing indicated that increased adrenergic input through the central amygdaloid nucleus-bone marrow axis drives abnormal myelopoiesis after TBI in a β2-adrenergic receptor-dependent manner, which is notably enhanced in aged mice after injury. Selective blockade of β2-adrenergic receptors rebalances abnormal myelopoiesis and improves the outcomes of aged mice after TBI. We therefore demonstrate that increased β2-adrenergic input-driven abnormal myelopoiesis exacerbates post-TBI neuroinflammation in the aged, representing a mechanism underlying the poorer recovery of aged patients and that blockade of β2-adrenergic receptor is a potential approach to promote neurological recovery after TBI.

Dysfunctional host cellular immune responses are associated with mortality in melioidosis

ABSTRACT Melioidosis is a tropical infection caused by the intracellular pathogen Burkholderia pseudomallei, an underreported and emerging global threat. As melioidosis-associated mortality is frequently high despite antibiotics, novel management strategies are critically needed. Therefore, we sought to determine whether functional changes in the host innate and adaptive immune responses are induced during acute melioidosis and are associated with outcome. Using a unique whole blood stimulation assay developed for use in resource-limited settings, we examined induced cellular functional and phenotypic changes in a cohort of patients with bacteremic melioidosis prospectively enrolled within 24 h of positive blood culture and followed for 28 days. Compared to healthy controls, melioidosis survivors generated an IL-17 response mediated by Th17 cells and terminally-differentiated effector memory CD8+ T cells (P < .05, both), persisting to 28 days after enrolment. Furthermore, melioidosis survivors developed polyfunctional cytokine production in CD8+ T cells (P < .01). Conversely, a reduction in CCR6+ CD4+ T cells was associated with higher mortality, even after adjustments for severity of illness (P = 0.004). Acute melioidosis was also associated with a profound acute impairment in monocyte function as stimulated cytokine responses were reduced in classical, intermediate and non-classical monocytes. Impaired monocyte cytokine function improved by 28-days after enrolment. These data suggest that IL-17 mediated cellular responses may be contributors to host defense during acute melioidosis, and that innate immune function may be impaired. These insights could provide novel targets for the development of therapies and vaccine targets in this frequently lethal disease.

Long-term changes in the phenotype and cytokine production of monocytes in COVID-19 recovered and vaccinated individuals.

Monocytes play a crucial role in the immune response against pathogens. Here, we sought to determine COVID-19 and the vaccine Gam-COVID-Vac induce long-term changes in the phenotype and cytokine production of circulating monocytes. Monocytes were purified from peripheral blood mononuclear cells of healthy donors who had not had COVID-19 or vaccination, who had received two doses of Gam-COVID-Vac, and who had mild/moderate COVID-19 in the last 6 months and evaluated by flow cytometry. To investigate the effect of SARS-CoV-2 proteins, monocytes were cultured for 2 days with or without stimulation with recombinant SARS-CoV-2 S1 and N peptides. Monocytes obtained from vaccinated and recovered individuals showed increased basal expression of HLA-DR, CD63, CXCR2, and TLR7. We also observed an increased frequency of CD63+ classical monocytes in both groups, as well as an increased frequency of HLA-DR+ non-classical monocytes in the COVID-19-recovered group compared to the control group. Monocytes from vaccinated and recovered donors produced higher basal levels of IL-6, IL-1β, and TNF-α cytokines. Ex vivo stimulation with SARS-CoV-2 antigens induced increased expression of HLA-DR and TLR7 on monocytes obtained from the control group. The challenge with SARS-CoV-2 antigens had no effect on the production of IL-6, IL-1β, and TNF-α cytokines by monocytes. The acquired data offer compelling evidence of enduring alterations in both the phenotype and functional status of circulating monocytes subsequent to vaccination with Gam-COVID-Vac and mild/moderate COVID-19 infection. At least some of these changes appear to be a consequence of exposure to SARS-CoV-2 S1 and N antigens.

Single-cell transcriptomics by clinical course of Mycobacterium avium complex pulmonary disease

Mycobacterium avium complex pulmonary disease (MAC-PD) has a heterogeneous clinical course. However, immune profiles associated with MAC-PD clinical course are limited. We performed single-cell RNA sequencing of peripheral blood mononuclear cells from 21 MAC-PD patients divided into three clinical courses: group A, spontaneous culture conversion; group B, stable disease without antibiotic treatment; and group C, progressive disease with antibiotic treatment. A lower proportion of NK cells and higher proportion of monocytes were noted in group C compared to combined groups A and B. The proportion of classical monocytes was higher in group C compared to groups A and B, while the proportion of non-classical monocytes decreased. EGR1, HSPA1A, HSPA1B, and CD83 were up-regulated in spontaneous culture conversion group A compared to progressive disease group C. Up-regulation of MYOM2 and LILRA4 and down-regulation of MT-ATP8, CD83, and CCL3L1 was found in progressive disease group C. PCBP1, FOS, RGCC, S100B, G0S2, AREG, and LYN were highly expressed in favorable treatment response compared to unfavorable response. Our findings may offer a comprehensive understanding of the host immune profiles that influence a particular MAC-PD clinical course and could suggest an immunological mechanism associated with the disease progression of MAC-PD.

Effects of increased nitrate intake from beetroot juice on blood markers of oxidative stress and inflammation in older adults with hypertension

BackgroundVascular oxidative stress and low-grade inflammation are important in the pathology of cardiovascular disorders, including hypertension. Cell culture and animal studies suggest that inorganic dietary nitrate may attenuate oxidative stress and inflammation through nitric oxide (NO), and there is a need to investigate whether this translates to humans. AimIn this randomised, placebo-controlled crossover study, by measuring a combination of multiple blood biomarkers, we evaluated whether previously reported benefits of dietary nitrate translate to a reduced oxidative stress and an improved inflammation status in 15 men and women (age range: 56–71 years) with treated hypertension. MethodsWe investigated the effects of a single ∼400 mg-dose of nitrate at 3 h post-ingestion (3H POST) and the daily consumption of 2 × ∼400 mg of nitrate over 4 weeks (4WK POST), through nitrate-rich versus nitrate-depleted (placebo) beetroot juice. Measurements included plasma nitrate and nitrite (NOx), oxidised low-density lipoprotein (oxLDL), F2-isoprostanes, protein carbonyls, oxidised (GSSG) and reduced glutathione (GSH); and serum high-sensitive C-reactive protein (hsCRP), chemokines, cytokines, and adhesion molecules. Flow cytometry was used to assess the relative proportion of blood monocyte subsets. ResultsAt 4WK POST nitrate intervention, the oxLDL/NOx ratio decreased (mainly due to increases in plasma nitrate and nitrite) and the GSH/GSSG ratio (a sensitive biomarker for alterations in the redox status) increased, compared with placebo (for both ratios P < 0.01). The relative proportion of classical (CD14+CD16−) monocytes decreased at 4WK POST for placebo compared to nitrate intervention (P < 0.05). Other oxidative stress and inflammatory markers were not altered by increased nitrate intake relative to placebo. ConclusionsThe data from this study point toward a subtle alteration in the redox balance toward a less pro-oxidative profile by a regular intake of inorganic nitrate from plant foods. Clinical trial registry numberNCT04584372 (ClinicialTrials.gov).

Immunophenotypic Evaluation of Circulating Monocyte Subsets in Beta Thalassemia Major Patients

Abstract Background β-thalassemia is a major public health problem in Egypt with particularly high incidence due to strong cultural preference for consanguineous marriages, and the high carrier rate. Iron overload, which is caused by increased gut iron absorption secondary to ineffective erythropoiesis, and regular blood transfusion is linked to accumulation within different organs with subsequent organ dysfunction. Monocytes are important lineages of innate immunity which activate the adaptive immune response and play a pivotal role in the host response to pathogens. Human blood monocyte subsets classification is based on the expression of CD14 and CD16 cell surface receptors. Aim of the Work To evaluate the circulating monocyte subsets in β-thalassemia major (β-TM) patients and its potential role in prediction of iron overload and chronic inflammation in β- thalassemia major patients. Patients and Methods A Case-Control study conducted on 40 previously diagnosed beta thalassemia major patients attending the outpatient clinic of Ain Shams University Pediatric Hospital, and 20 age and sex matched healthy control subjects. CBC, iron profile, CRP and immunophenotyping for monocyte subsets classification were done. Results Non classical monocytes were found statistically significantly higher in thalassemic patients especially in those with serum ferritin level &amp;lt; 1000 ng/ml (P value &amp;lt; 0.01). In addition, classical monocytes were found to be statistically significantly lower among the patients’ group (P value &amp;lt; 0.01). A negative correlation was found between intermediate monocytes and Transferrin saturation percent (TS%) suggesting its protective role in iron overload thalassemia patients. By using ROC curve, non- classical monocytes predicted ferritin &amp;lt; 1000 ng/ml at a cutoff point of 17.8 % and 0.05 x 10∧9/L for relative and absolute counts respectively. Conclusion All monocyte subsets proposed to have role in predicting iron overload and chronic inflammation β-TM patients. We recommend further studies on β-TM patients with the engagement of activity assays of monocyte subsets to clarify the co-regulatory role in iron hemostasis and chronic inflammation.

P-306 GnRH agonists exert a positive influence on regulatory immune cells in the endometrium of women with adenomyosis

Abstract Study question How does treatment with GnRH-agonist (GnRH-a) prior to frozen embryo transfer (FET) in women with adenomyosis affect immune cell populations in endometrium and peripheral blood? Summary answer GnRH-a induces a decrease in monocytes and an increase in CD4+T-cells in peripheral blood and a decrease classical monocytes and T-regulatory cells in the endometrium What is known already Adenomyosis is associated with lower implantation and higher miscarriage rates. Observational studies have reported improved pregnancy outcomes in women with adenomyosis after assisted reproductive technologies (ART) and pre-treatment with GnRH-a prior to FET. However, the specific mechanisms of GnRHa action beyond central hormonal suppression remain unclear, but may be mediated through local and/or systemic immunomodulation. Study design, size, duration Prospective cohort study including infertile women with adenomyosis and GnRH-a treatment ≥3 months prior to FET. Pre- and post-treatment analysis of blood and endometrium samples were compared. Recruitment is ongoing with n = 30 women included thus far, 16 of whom have completed the study, while the remaining are still undergoing GnRH-a therapy. Participants/materials, setting, methods Adenomyosis was diagnosed sonographically if ≥ 2 criteria published by the MUSA group were met. Peripheral blood and endometrial samples were obtained before and after treatment with GnRH-a. Plasma cells and uterine Natural Killer (uNK) cells in endometrial biopsies were quantified with immunhistochemistry (IHC), and leukocyte populations in all samples were analysed using flow cytometry (FC). Pre- and post-treatment levels of immune cells and percentage of leukocyte populations were compared using Wilcoxon rank-sum tests (p &amp;lt; 0.05 significant) Main results and the role of chance Interim analysis of immune cell populations in peripheral blood and endometrium of the 16 women who already completed the study was performed. At baseline, IHC showed elevated plasma cell concentrations indicating chronic endometritis in 3/30 patients (10%), while uNK cells were elevated (&amp;gt;300/mm2) in 13/30 patients (43.3%). FC analysis of peripheral blood showed a significant decrease in percentage of monocytes (p = 0.01) and increase in percentage of T-cells (p = 0.03), specifically concentration of CD4+T-cells (p = 0.03), after treatment with GnRH-a as compared to pre-treatment levels. In the endometrium, following GnRH-a therapy, we observed a decrease in both the percentage of classical monocytes (p = 0.03) and T-regulatory cells (p = 0.03). All 16 participants who completed the study already underwent FET, resulting in ten pregnancies (62.5%), although four resulted in miscarriage, while the outcome of the remaining pregnancies is still pending. Limitations, reasons for caution These are preliminary results whose generalizability needs to be confirmed in a larger study population. No correction for multiple comparisons was performed and significant findings have to be interpreted with caution. Wider implications of the findings Elevated uNK on IHC cells seen at baseline may be associated with implantation failure, miscarriage. CD4+ and DN T-cells are important mediators of reproductive immune tolerance. In women with adenomyosis, GnRH-a may have a positive impact on these regulatory immune cells in peripheral blood as well as in the endometrium. Trial registration number Not applicable

A pilot study of monocytes in relapsing remitting multiple sclerosis: Correlation with disease activity.

Background: Numerous immune cells are involved in developing multiple sclerosis (MS). Monocytes are believed to be the first to enter the brain and initiate inflammation. The role of monocyte subtypes in MS needs to be better understood. Objective: The current study aims to investigate the presence of different subsets of monocytes in relapsing-remitting MS (RRMS) Egyptian patients and their correlation with disease activity. Methods: This study included 44 RRMS patients (22 patients in relapse, 22 patients in remission), diagnosed according to the 2017 MacDonalds criteria, and 44 matched healthy controls. Personal and medical histories were taken from the patients, and the Expanded Disability Status Scale (EDSS) was used to evaluate the degree of impairment. Characterization of peripheral blood monocyte subsets was done by flow cytometry for all participants. Results: The percentage of classical, intermediate, and non-classical monocyte subsets showed a significant increase in RRMS patients than controls with p-values of 0.029, 0.049, and 0.043, respectively. In the RRMS patients, there were no statistically significant correlations (p-values >0.05) between the EDSS scores, the duration of disease, and number of relapses in the past year and the percentages of the various monocyte subsets. Furthermore, there were no significant differences in the percentage of each monocyte subset between RRMS patients in remission and those experiencing a relapse (p-values >0.05). However, patients with evidence of activity in magnetic resonance imaging (MRI) had a significantly high percentage of non-classical monocytes with a p-value of 0.002. Conclusion: In RRMS patients, the three monocyte subsets (classical, non-classical and intermediate) increase significantly regardless of the disease activity. This increase denotes the vital role of monocytes and innate immunity in MS pathology, especially the non-classical monocyte subset. These findings suggest that monocytes might be a promising MS therapeutic target.

Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

There is no treatment for acute aortic dissection (AAD) targeting inflammatory cells. We aimed to identify the new therapeutic targets associated with inflammatory cells. We characterized the specific distribution of myeloid cells of both human type A AAD samples and a murine AAD model generated using angiotensin II (ANGII) and β-aminopropionitrile (BAPN) by single-cell RNA sequencing (scRNA-seq). We also examined the effect of an anti-interleukin-1β (IL-1β) antibody in the murine AAD model. IL1B+ inflammatory macrophages and classical monocytes were increased in human AAD samples. Trajectory analysis demonstrated that IL1B+ inflammatory macrophages differentiated from S100A8/9/12+ classical monocytes uniquely observed in the aorta of AAD. We found increased infiltration of neutrophils and monocytes with the expression of inflammatory cytokines in the aorta and accumulation of inflammatory macrophages before the onset of macroscopic AAD in the murine AAD model. In blocking experiments using an anti-IL-1β antibody, it improved survival of murine AAD model by preventing elastin degradation. We observed the accumulation of inflammatory macrophages expressing IL-1β in both human AAD samples and in a murine AAD model. Anti-IL-1β antibody could improve the mortality rate in mice, suggesting that it may be a treatment option for AAD.

Causal role of immune cells on cervical cancer onset revealed by two-sample Mendelian randomization study

Cervical cancer (CC) is a prevalent gynecological cancer worldwide that significantly impacts the quality of life and the physical and mental well-being of women. However, there have been limited studies utilizing Mendelian randomization (MR) analysis to investigate the connection between immune cells and CC. This study is to investigate the causal effects of immune traits on CC and non-neoplastic conditions of the cervix. The GWAS data for 731 immunophenotypes and six GWAS data for CC from the FinnGen database were downloaded. Subsequently, a two-sample MR analysis was conducted using the MR Egger, Weighted median, Inverse variance weighted (IVW), Simple mode, and Weighted mode methods. Our study has identified the potential causal effects of immune traits on inflammatory diseases of the cervix, other noninflammatory disorders of the cervix uteri, carcinoma in situ of cervix uteri, adenocarcinomas of cervix, squamous cell neoplasms and carcinoma of cervix, as well as malignant neoplasm of the cervix uteri, with the respective numbers being 8, 6, 11, 8, 23, and 12, respectively. A strong correlation between classic monocytes and various cervical diseases was revealed. Furthermore, we discovered that B cells expressing BAFF-R have the ability to impede the advancement of malignant CC, specifically squamous cell neoplasms and carcinoma of cervix. Our study has demonstrated a significant association between immune traits and both CC and non-neoplastic conditions of the cervix through two-sample Mendelian randomization, providing valuable insights for future clinical research.

Single Cell Analysis Reveals Novel Immune Perturbations in Fibrotic Hypersensitivity Pneumonitis.

Rationale: Fibrotic hypersensitivity pneumonitis is a debilitating interstitial lung disease driven by incompletely understood immune mechanisms. Objectives: To elucidate immune aberrations in fibrotic hypersensitivity pneumonitis in single-cell resolution. Methods: Single-cell 5' RNA sequencing was conducted on peripheral blood mononuclear cells and bronchoalveolar lavage cells obtained from 45 patients with fibrotic hypersensitivity pneumonitis, 63 idiopathic pulmonary fibrosis, 4 non-fibrotic hypersensitivity pneumonitis, and 36 healthy controls in the United States and Mexico. Analyses included differential gene expression (Seurat), transcription factor activity imputation (DoRothEA-VIPER), and trajectory analyses (Monocle3/Velocyto-scVelo-CellRank). Measurements and Main Results: Overall, 501,534 peripheral blood mononuclear cells from 110 patients and controls and 88,336 bronchoalveolar lavage cells from 19 patients were profiled. Compared to controls, fibrotic hypersensitivity pneumonitis has elevated classical monocytes (adjusted-p=2.5e-3) and are enriched in CCL3hi/CCL4hi and S100Ahi classical monocytes (adjusted-p<2.2e-16). Trajectory analyses demonstrate that S100Ahi classical monocytes differentiate into SPP1hi lung macrophages associated with fibrosis. Compared to both controls and idiopathic pulmonary fibrosis, fibrotic hypersensitivity pneumonitis patient cells are significantly enriched in GZMhi cytotoxic T cells. These cells exhibit transcription factor activities indicative of TGFβ and TNFα/NFκB pathways. These results are publicly available at https://ildimmunecellatlas.org. Conclusions: Single-cell transcriptomics of fibrotic hypersensitivity pneumonitis patients uncovered novel immune perturbations, including previously undescribed increases in GZMhi cytotoxic CD4+ and CD8+ T cells - reflecting this disease's unique inflammatory T-cell driven nature - as well as increased S100Ahi and CCL3hi/CCL4hi classical monocytes also observed in idiopathic pulmonary fibrosis. Both cell populations may guide the development of new biomarkers and therapeutic interventions.

VNN2-expressing circulating monocytes exhibit unique functional characteristics and are decreased in patients with primary Sjögren's syndrome

ObjectiveThis study aims to elucidate the significance of VNN2 expression in peripheral blood monocytes and its clinical relevance in primary Sjögren's syndrome (pSS). MethodsWe investigated VNN2 expression by analyzing single-cell RNA sequencing (scRNA-seq) data from peripheral blood mononuclear cells. Flow cytometry was used to detect and compare VNN2 expression in total monocytes, classical monocytes (cMo), intermediate monocytes (iMo) and non-classical monocytes (ncMo). Additionally, we examined the expression of HLA, ICAM1, CD62L, ITGAM, S100A8, S100A9, CCR2, CCR6, CX3CR1 and CXCR3 in VNN2+ and VNN2- cells. We analyzed the correlation between VNN2 expression and clinical indicators and assessed the clinical utility of VNN2+ monocytes in pSS diagnosis using receiver operating characteristic curves. ResultsWe observed high VNN2 expression in monocytes, with significantly higher levels in CD14++ monocytes compared to ncMo. VNN2+ monocytes exhibited decreased expression of HLA and CD62L and increased expression of ICAM1, ITGAM, S100A8, S100A9, CCR2, CCR6, CX3CR1 and CXCR3 compared to VNN2- monocytes. Although scRNA-seq data showed that VNN2 mRNA was upregulated, cell surface expression of VNN2 was decreased in monocytes from pSS patients compared to healthy controls. The reduced levels of VNN2+ monocyte subpopulations in pSS patients were negatively correlated with anti-ribosome antibody levels and positively correlated with complement 4 levels. Detection of VNN2 expression in monocytes can aid in the auxiliary diagnosis of pSS. ConclusionMonocytes expressing cell surface VNN2 are significantly reduced in pSS patients. This suggests a potential role for VNN2 in pSS development and its potential use as a diagnostic marker for pSS.

Disease activity drives divergent epigenetic and transcriptomic reprogramming of monocyte subpopulations in systemic lupus erythematosus

ObjectivesSystemic lupus erythematosus (SLE) is characterised by systemic inflammation involving various immune cell types. Monocytes, pivotal in promoting and regulating inflammation in SLE, differentiate from classic monocytes into intermediate and...

Heme-oxygenase-1: A potential biomarker for ICIs in patients with advanced lung cancer.

e20505 Background: Immune checkpoint inhibitors (ICIs) significantly improved the outcome in patients with lung cancer. However, heterogeneity in clinical benefit and absence of a reliable biomarker still represent an unmet need. Metabolic reprogramming plays a critical role in cancer behaviour. In particular, heme-ossigenase-1 (HO-1) expression on monocytes may have a role in treatment resistance. Methods: “ImmunoEGA” is a prospective, observational study in patients with advanced tumors, treated with ICIs at the ‘Maggiore della Carità’ Hospital in Novara, IT. Overall, 150 patients have been recruited from 2018 to 2023; of these, 122 were affected by lung cancer. In the present study we analysed monocytes subpopulations and HO-1 expression on blood samples collected from 34 patients at the following time points: baseline, at time of disease evaluation and at disease progression (PD). Monocytes were classified into three different phenotypes based on CD14 and CD16 expression: classical (CD14+CD16–), non-classical (CD14-CD16+) and intermediate (CD14+CD16+). The primary endpoint was treatment outcome in terms of progression free survival (PFS) and/or overall survival (OS). Results: At baseline, the frequency of classical monocytes was higher as compared to non-classical and intermediate subgroups, independently from type of response (mean percentage: 48,4% vs 7,8 vs 23,9%; p &lt; 0.0001). Compared with baseline, we observed a relative increase in the frequency of classical (54,8 vs. 56,9) and intermediate (19,5 vs. 21,1) monocytes in patients with stable disease (SD) at the time of best response. Conversely, patients with PD showed an increase in non-classical (6,3 vs.14,1) and intermediate monocytes (26,3 vs. 30,9). In patients with partial response (PR) all monocyte subgroups increased proportionally (classical: 40,6 vs. 45,5; intermediate 24,2 vs. 25,9; non-classical 7,8 vs. 13,6). A higher baseline frequency of classical monocyte positively correlated with PFS (14 vs 6.5 m, p = 0,0171); these data were not observed for OS. No differences were detectable in HO-1 expression across monocytes subtypes at baseline and at the time of best response (SD or PR). Conversely, HO-1 expression showed an increasing trend across monocytes subpopulations at PD. Lower on classical (mPFS 15 vs 7 m, p = 0,0186) and intermediate monocytes (mPFS 15.5 vs 6.5 m p = 0,0057) at baseline correlated with better PFS. Patients with lower HO-1 expression on intermediate monocytes at baseline had a better outcome (OS 18 vs 11.5 m, p = 0.0378). Conclusions: In patients with lung cancer, higher frequency of classical monocytes and lower HO-1 expression in intermediate and classical monocytes predict better outcomes with ICI. The expression of HO-1 in classical and intermediate monocytes significantly increases at PD, suggesting its implication in promoting cancer progression. HO-1 expression could be used as a biomarker of immunotherapy benefit.

CD4 effector T cell expansion to identify objective responses to the CD40 agonist mitazalimab in combination with modified FOLFIRINOX (mFFX) as first-line therapy for metastatic pancreatic ductal adenocarcinoma (mPDAC) in the OPTIMIZE-1 study.

2569 Background: CD40 agonists have the potential to enhance the efficacy of standard of care chemotherapy and to trigger antitumor immunity. Preclinical models indicate that the success of this approach depends on appropriate sequencing of chemotherapy with a CD40 agonist. Preliminary efficacy results from the OPTIMIZE-1 Phase II study (NCT04888312) combining mitazalimab (anti-CD40) with mFFX chemotherapy in patients with mPDAC have been previously reported. Here, we investigated immunological determinants associated with favorable outcomes with this combination therapy. Methods: Newly diagnosed, chemotherapy-naive patients with mPDAC received mitazalimab (900 µg/kg) on day 1 prior to beginning an every 2-week mFFX regimen on day 8, followed by mitazalimab 48 hours later. Tumor response was determined using RECIST v1.1 criteria. Peripheral blood was analyzed for selected cytokines and chemokines and by flow cytometry to assess changes in leukocyte subsets including B cells, monocytes, T cells, and NK cells. Flow cytometric data from evaluable patients were subjected to unsupervised hierarchical clustering to identify immune subsets. A classifier was generated from an interim dataset (n=21) using a random forest model to predict cell populations and their association with tumor response, which were then applied to the full dataset (n=47). Results: Mitazalimab triggered an expected immune response characterized by transient cytokine (IFNg and MCP-1) release and B cell depletion. Chemotherapy caused a reduction in classical monocytes and proliferating CD4+ T cells. Both mitazalimab and chemotherapy caused a transient reduction in circulating dendritic cells and NK cells. Tumor response was associated with an expansion in the frequency of effector CD4 T cells (p &lt; 0.0001) at day 8 after receiving mitazalimab. In a blinded analysis based on the classifier, CD4 T cell expansion was linked to an early tumor response to treatment (accuracy 70%, sensitivity 68%, specificity 72%). Conclusions: Mitazalimab and mFFX differentially modulate immune responses in patients with mPDAC. Pharmacological analyses identify mitazalimab-induced expansion of CD4 effector T cells one week after first administration as a correlate of treatment outcomes. These data suggest the contribution of mitazalimab to tumor responses, and further substantiate a priming dose of mitazalimab, prior to administering chemotherapy.

Within- and between-subject biological variation estimates for the enumeration of lymphocyte deep immunophenotyping and monocyte subsets.

This study aimed to deliver biological variation (BV) estimates for 25 types of lymphocyte subpopulations subjected to deep immunophenotyping (memory T/B cells, regulatory T cells, etc.) and classical, intermediate, and nonclassical monocyte subsets based on the full spectrum flow cytometry (FS-FCM) and a Biological Variation Data Critical Appraisal Checklist (BIVAC) design. Samples were collected biweekly from 60 healthy Chinese adults over 10 consecutive two-week periods. Each sample was measured in duplicate within a single run for lymphocyte deep immunophenotyping and monocyte subset determination using FS-FCM, including the percentage (%) and absolute count (cells/μL). After trend adjustment, a Bayesian model was applied to deliver the within-subject BV (CVI) and between-subject BV (CVG) estimates with 95 % credibility intervals. Enumeration (% and cells/μL) for 25 types of lymphocyte deep immunophenotyping and three types of monocyte subset percentages showed considerable variability in terms of CVI and CVG. CVI ranged from 4.23 to 47.47 %. Additionally, CVG ranged between 10.32 and 101.30 %, except for CD4+ effector memory T cells re-expressing CD45RA. No significant differences were found between males and females for CVI and CVG estimates. Nevertheless, the CVGs of PD-1+ T cells (%) may be higher in females than males. Based on the desired analytical performance specification, the maximum allowable imprecision immune parameter was the CD8+PD-1+ T cell (cells/μL), with 23.7 %. This is the first study delivering BV estimates for 25 types of lymphocyte subpopulations subjected to deep immunophenotyping, along with classical, intermediate, and nonclassical monocyte subsets, using FS-FCM and adhering to the BIVAC design.