Circulating Leukocyte Populations Research Articles

#4024 PHASE 1 STUDY OF THE SAFETY, TOLERABILITY, PHARMACOKINETICS, AND PHARMACODYNAMICS OF POVETACICEPT FOR AUTOIMMUNE GLOMERULONEPHRITIDES

Abstract Background and Aims B cell activating factor (BAFF) of the tumor necrosis factor (TNF) family and a proliferation-inducing ligand (APRIL), cytokines which bind and signal through BAFF-R, transmembrane activator and CAML interactor (TACI), and/or B cell maturation antigen (BCMA) on B cells, play overlapping and non-redundant roles in B cell development, proliferation, function, and survival. Therapeutic agents targeting BAFF and/or APRIL have demonstrated promising clinical potential in autoantibody-related glomerulonephritides (GN) such as lupus nephritis (LN), immunoglobulin (Ig) A nephropathy (IgAN), membranous nephropathy, and other B cell-related diseases such as systemic lupus erythematosus (SLE); however, there is still need for more safe and efficacious therapies. Povetacicept (ALPN-303) is an Fc fusion protein of an engineered TACI variant TNFRSF domain (vTD) with enhanced affinity for APRIL and BAFF which mediates more potent inhibitory activity than wild type (WT) TACI-Fc or BAFF- or APRIL-specific antibodies. In preclinical studies, povetacicept demonstrated enhanced pharmacokinetic (PK) and immunomodulatory properties vs. WT TACI-Fc, which may translate to lower and/or less frequent doses in humans. Povetacicept also suppressed autoantibodies, renal IgG deposition, and nephritis in mouse models. Povetacicept may therefore significantly improve clinical outcomes in autoantibody-mediated GNs and other B cell-related diseases. Method In this first-in-human study (NCT05034484), 66 healthy adult volunteers were randomized 4:2 into single ascending dose cohorts of intravenous (IV) or subcutaneous (SC) povetacicept or placebo. Participants were followed to assess safety and PK, circulating Ig, galactose-deficient IgA1 (Gd-IgA1), and circulating leukocyte populations. Results Povetacicept has been well tolerated in all cohorts evaluated as single IV or SC doses of up to 960 mg. Overall, it exhibits dose-related PK and expected pharmacodynamic (PD) effects, including dose-related reductions in serum IgA, IgM, IgG, and Gd-IgA1 (Figure 1), and in circulating antibody-secreting cells (ASC; plasmablasts and plasma cells) (Figure 2). In the same setting, these PD effects appear greater than those reported for WT TACI-Fc molecules and appear to be saturated at doses ≥80 mg. Coverage of free APRIL was maintained for 2-3 weeks with 80 mg and ≥4 weeks with 240 mg, respectively. The most frequent adverse event has been mild headache. To date, there have been no imbalances of infections between placebo and povetacicept groups, no treatment-related reactions other than mild injection site pain, and no adverse trends in safety laboratories. Conclusion To date, povetacicept has demonstrated acceptable safety and tolerability as single IV or SC doses, exhibiting dose-dependent PK and PD that appear to differentiate favorably vs WT TACI-Fc. Based on the magnitude and duration of the observed PD effects, dose regimens of 80-240 mg SC every 4 weeks are anticipated for use in future studies. Overall, the study findings support future clinical development of povetacicept in multiple autoantibody-related GN, as well as other B cell- and/or autoantibody-related diseases such as SLE and autoantibody-associated cytopenias.

Systemic Inflammatory Response as a Prognostic Factor in Breast Cancer. Part I. Tumor-Promoting Inflammation. Serum Inflammatory Markers

Chronic inflammation caused by exposure to external or internal factors increases the risk of developing malignancies and promotes tumor progression due to the influence on the key elements of carcinogenic mechanisms. At the system level signs of a chronic inflammation are manifested by an increase of inflammatory mediators and acute phase proteins levels in the blood, a change in the ratio of circulating leukocyte populations, and disturbances in the hemostasis system. This review is devoted to serum and hematological parameters of the systemic inflammatory response (SIR) in breast cancer (BC). The first part of the review outlines general concept about the role of inflammatory factors in the development of malignant tumors. It provides information on the most well studied serum inflammatory markers in breast cancer: cytokines, including interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-), as well as C-reactive protein (CRP). The main properties of these polypeptides, which link them with tumor-promoting inflammation, are considered. An analysis of the data on the clinical significance of the serum level of cytokines and CRP in breast cancer accumulated to date is presented. Correlations of the elevated levels of the serum inflammatory markers with clinical and morphological characteristics of the disease, tumor response to chemotherapy, overall and relapse-free survival of patients indicate the feasibility of in-depth investigation of the issue for the purpose of the practical application of the systemic inflammatory markers as predictive and prognostic indicators in BC.

Cellular immunophenotype of major spine surgery in adults.

ASD reconstructions are a major, sterile traumatic insult, likely causing perturbations to the immune systems. The immune response to surgery is associated with outcomes. The purpose of this study was to examine for a detectable immune signature associated with ASD surgery. Consecutive patients undergoing ASD surgery were approached and enrolled. Peripheral blood was drawn before incision, 4h after, and 24h after incision. Blood was stabilized and comprehensive flow cytometric immunophenotyping performed. Leukocyte population frequency, absolute number and activation marker expression were defined. Immunologic features were defined and analyzed by hierarchical clustering and principal component analysis (PCA). Changes over time were evaluated by repeated measures ANOVA (RMANOVA) and were corrected for a 1% false discovery rate. Post hoc testing was by Dunn's test. p values of < = 0.05 were considered significant. Thirteen patients were enrolled; 11(85%) F, 65.4years (± 7.5), surgical duration 418 ± 83min, EBL 1928 ± 1253mL. Hierarchical clustering and PCA found consistent time from incision-dependent changes. HLA-DR and activating co-stimulatory molecule CD86 were depressed at 4h and furthermore at 24h on monocyte surfaces. CD4 + HLA-DR + T cells, but not CD8 +, increased over time with increased expression of PD-1 at 4 and 24h. Despite surgery and patient heterogeneity, we identified an immune signature associated with the sterile trauma of ASD surgery. Circulating leukocyte populations change in composition and signaling protein expression after incision and persisting to 24h after incision, suggesting an immunocompromised state. Further work may determine relationships between this state and poor outcomes after surgery.

Absence of CD4+ and CD8+ T cell expansion after primary multimodal treatment predicts early progression in inoperable stage III NSCLC.

e20590 Background: There are no blood-based biomarkers for survival prediction in inoperable stage III NSCLC patients. We propose a method for calculation of novel area under curve (AUC) biomarkers of the dynamic change of multiple leukocyte subpopulations before, during, and after thoracic irradiation (TRT), chemoradiotherapy (CRT), and chemo-radio-immunotherapy (CRT-ICI) in this patient cohort. The extracted biomarkers identify patients with early progression after TRT. Methods: 20 patients (17 male, 3 female), at median age of 65.5 years (range 34 to 79) were enrolled in the study. The median follow-up time was 60 weeks. Eleven patients suffered from adenocarcinoma, 8 squamous cell carcinoma, and 1 undifferentiated NSCLC. They received TRT (2/20, 10%), CRT (11/20, 55%), or CRT-ICI (7/20, 35%). One patient (1/20, 5%) withdrew consent and was excluded from analysis. Primary endpoints were progression free survival (PFS) at 6 and 12 months. Patient blood was analyzed via flow cytometry for 7 circulating leukocyte populations at baseline, twice during radiotherapy, at the end of radiotherapy (RTend), and 10, 20, 35, 48, and 60 weeks after enrollment. We analyzed CD3+ total T cells, CD4+ T cells, CD8+ T cells, CD19+/CD20+ B cells, CD3-CD56+ NK cells, CD56 bright NK cells, and CD56 dim NK cells. Here, we report on CD4+ and CD8+ T cells. The AUC from RTend to zenith of absolute cell counts provided aggregate measures for the time-course data. We performed hierarchical clustering and cluster characterization. Relevant features were selected by stepwise drop-out. Results: Clustering of the AUC between RTend and zenith for CD4+ T cells and CD8+ T cells delineated two prognostic groups. The favorable group was characterized by higher AUC values for CD4+ and CD8+ T cells compared to the unfavorable group. All patients (9/9, 100%) in the favorable group versus 36.4% (4/11) patients in the unfavorable group were progression-free at 6 months (Fisher´s exact test, two-tailed: p-value = 0.00472). This effect was observed as a trend with PFS at 12 months. Here 66.6% (6/9) of patients had PFS at 12 months in the favorable group versus 27.3% (3/11) in the unfavorable group (Fisher´s exact test, two-tailed: p-value = 0.175). There is a directly proportional relationship of the reported AUC values to PFS at 6 months and 12 months. Conclusions: Patients who responded with T cell expansion after immunogenic cell death by TRT, CRT, or CRT-ICI had significantly longer PFS compared to those without increase. Longitudinal monitoring of CD4+ and CD8+ T cells and the AUC from RTend to zenith is a promising biomarker for detecting early progression in the present study which is the subject of validation in an ongoing prospective study (PRECISION, NCT05027165).

Peripheral Blood Mononuclear Cell Populations Correlate with Outcome in Patients with Metastatic Breast Cancer.

Local tumor-associated immune cells hold prognostic and predictive value in various forms of malignancy. The role of systemic, circulating leukocytes is, however, not well-characterized. In this prospective and explorative study, we aim to delineate the clinical relevance of a broad panel of circulating immune cells in 32 patients with newly diagnosed metastatic breast cancer (MBC) before the start of systemic treatment. Freshly isolated peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry and evaluated for potential associations to clinicopathological variables and patient outcome. We show that the levels of specific circulating leukocyte populations are associated with clinical parameters such as hormone receptor status, histological subtype, number of circulating tumor cells (CTCs) and metastatic burden. Importantly, high levels of CD8+ cytotoxic T lymphocytes (CTLs) are significantly linked to improved overall survival (OS). In patients with estrogen receptor (ER)-positive primary tumors, high levels of circulating CTLs and non-classical (CD14+CD16++) monocytes were associated with improved OS, whereas in patients with ER-negative tumors low levels of circulating natural killer (NK) cells potentially associate with improved OS. We propose that the levels of specific circulating immune cell populations, such as CD8+ CTLs, may be used to predict clinical outcomes in MBC patients. Thus, larger studies are warranted to validate these findings.

Recruitment of \u03b14\u03b27 monocytes and neutrophils to the brain in experimental colitis is associated with elevated cytokines and anxiety-like behavior

BackgroundBehavioral comorbidities, such as anxiety and depression, are a prominent feature of IBD. The signals from the inflamed gut that cause changes in the brain leading to these behavioral comorbidities remain to be fully elucidated. We tested the hypothesis that enhanced leukocyte–cerebral endothelial cell interactions occur in the brain in experimental colitis, mediated by α4β7 integrin, to initiate neuroimmune activation and anxiety-like behavior.MethodsFemale mice treated with dextran sodium sulfate were studied at the peak of acute colitis. Circulating leukocyte populations were determined using flow cytometry. Leukocyte–cerebral endothelial cell interactions were examined using intravital microscopy in mice treated with anti-integrin antibodies. Brain cytokine and chemokines were assessed using a multiplex assay in animals treated with anti-α4β7 integrin. Anxiety-like behavior was assessed using an elevated plus maze in animals after treatment with an intracerebroventricular injection of interleukin 1 receptor antagonist.ResultsThe proportion of classical monocytes expressing α4β7 integrin was increased in peripheral blood of mice with colitis. An increase in the number of rolling and adherent leukocytes on cerebral endothelial cells was observed, the majority of which were neutrophils. Treatment with anti-α4β7 integrin significantly reduced the number of rolling leukocytes. After anti-Ly6C treatment to deplete monocytes, the number of rolling and adhering neutrophils was significantly reduced in mice with colitis. Interleukin-1β and CCL2 levels were elevated in the brain and treatment with anti-α4β7 significantly reduced them. Enhanced anxiety-like behavior in mice with colitis was reversed by treatment with interleukin 1 receptor antagonist.ConclusionsIn experimental colitis, α4β7 integrin-expressing monocytes direct the recruitment of neutrophils to the cerebral vasculature, leading to elevated cytokine levels. Increased interleukin-1β mediates anxiety-like behavior.

Maternal body mass index is associated with an altered immunological profile at 28 weeks of gestation.

Healthy pregnancy is accompanied by various immunological and metabolic adaptations. Maternal obesity has been implicated in adverse pregnancy outcomes such as miscarriage, preeclampsia, and gestational diabetes mellitus (GDM), while posing a risk to the neonate. There is a lack of knowledge surrounding obesity and the maternal immune system. The objective of this study was to consider if immunological changes in pregnancy are influenced by maternal obesity. Peripheral blood was collected from fasted GDM-negative pregnant women at 26-28 weeks of gestation. Analysis was done using immunoassay, flow cytometry, bioenergetics analysis, and cell culture. The plasma profile was significantly altered with increasing BMI, specifically leptin (r = 0.7635), MCP-1 (r = 0.3024), and IL-6 (r = 0.4985). Circulating leukocyte populations were also affected with changes in the relative abundance of intermediate monocytes (r = -0.2394), CD4:CD8 T-cell ratios (r = 0.2789), and NKT cells (r = -0.2842). Monocytes analysed in more detail revealed elevated CCR2 expression and decreased mitochondrial content with increased BMI. However, LPS-stimulated cytokine production and bioenergetic profile of PBMCs were not affected by maternal BMI. The Th profile skews towards Th17 with increasing BMI; Th2 (r = -0.3202) and Th9 (r = -0.3205) cells were diminished in maternal obesity, and CytoStim™-stimulation exacerbates IL-6 (r = 0.4166), IL-17A (r = 0.2753), IL-17F (r = 0.2973), and IL-22 (r = 0.2257) production with BMI, while decreasing IL-4 (r = -0.2806). Maternal obesity during pregnancy creates an inflammatory microenvironment. Successful pregnancy requires Th2-biased responses yet increasing maternal BMI favours a Th17 response that could be detrimental to pregnancy. Further research should investigate key populations of cells identified here to further understand the immunological challenges that beset pregnant women with obesity.

Treatment With the CSF1R Antagonist GW2580, Sensitizes Microglia to Reactive Oxygen Species.

Microglia activation and proliferation are hallmarks of many neurodegenerative disorders and may contribute to disease pathogenesis. Neurons actively regulate microglia survival and function, in part by secreting the microglia mitogen interleukin (IL)-34. Both IL-34 and colony stimulating factor (CSF)-1 bind colony stimulating factor receptor (CSFR)1 expressed on microglia. Systemic treatment with central nervous system (CNS) penetrant, CSFR1 antagonists, results in microglia death in a dose dependent matter, while others, such as GW2580, suppress activation during disease states without altering viability. However, it is not known how treatment with non-penetrant CSF1R antagonists, such as GW2580, affect the normal physiology of microglia. To determine how GW2580 affects microglia function, C57BL/6J mice were orally gavaged with vehicle or GW2580 (80mg/kg/d) for 8 days. Body weights and burrowing behavior were measured throughout the experiment. The effects of GW2580 on circulating leukocyte populations, brain microglia morphology, and the transcriptome of magnetically isolated adult brain microglia were determined. Body weights, burrowing behavior, and circulating leukocytes were not affected by treatment. Analysis of Iba-1 stained brain microglia indicated that GW2580 treatment altered morphology, but not cell number. Analysis of RNA-sequencing data indicated that genes related to reactive oxygen species (ROS) regulation and survival were suppressed by treatment. Treatment of primary microglia cultures with GW2580 resulted in a dose-dependent reduction in viability only when the cells were concurrently treated with LPS, an inducer of ROS. Pre-treatment with the ROS inhibitor, YCG063, blocked treatment induced reductions in viability. Finally, GW2580 sensitized microglia to hydrogen peroxide induced cell death. Together, these data suggest that partial CSF1R antagonism may render microglia more susceptible to reactive oxygen and nitrogen species.

225 The role of PCγ2 in the in vitro activation of neutrophils by type VII collagen immune complexes

Phospholipase Cγ2 (PLCγ2) is a protein expressed predominantly in hematopoietic cells and has a great importance in immunoreceptor signaling. Our group previously found that PLCγ2 deficient mice were protected from autoantibody-mediated skin blistering model induced by the systemic injection of antibodies against type VII collagen (C7), which is located in the basement membrane. The inflammation mediated mainly by neutrophils was completely abrogated in PLCγ2 deficient mice, but the mechanism is presently unknown. We aimed to examine the expression of PLCγ2 in the main circulating leukocyte populations and investigate the in vitro activation of PLCγ2 deficient neutrophils by C7-anti-C7 immune complexes. Expression of PLCγ2 in circulating leukocyte populations was measured by flow cytometry after intracellular labelling. We used bone marrow derived neutrophils to examine in vitro cell activation plated onto immobilized C7-anti-C7 immune complex surfaces. Type VII collagen or anti-C7 served as control surfaces. Cell spreading was evaluated using phase-contrast microscopy. Production of reactive oxygen species (ROS) was measured in a cytochrome-C based reduction assay. PLCγ2 was present in neutrophils, eosinophils, monocytes and B cells but not in T cells of the wild type mice. While examining the effector functions of neutrophils activated by C7-anti-C7 immune complexes, we found that nearly a third of the cells of the wild type mice spread and a great amount of ROS was produced. On the contrary, both neutrophil spreading and ROS production were completely absent in immune complex-treated PLCγ2 deficient cells. Our results show that the PLCγ2 is expressed in the major myeloid cell populations-expression of the circulating immune cells and plays a crucial role in the spreading and ROS-production of neutrophils activated by C7-anti-C7 immune complexes in vitro, which can explain why PLCγ2 deficient mice are protected in autoantibody mediated skin blistering.

FC 037NEUTROPHILS PLAY A KEY ROLE IN THE INITIATION OF GLOMERULAR HEMATURIA IN A POSTINFECTIOUS IGAN EXPERIMENTAL MODEL

Abstract Background and Aims Hematuria is a common finding in patients with IgA nephropathy (IgAN), occurring mainly after upper respiratory tract infections. Hematuria can lead to acute kidney injury and chronic loss of renal function in IgAN. However, the mechanisms involved in egression of erythrocytes from the glomerular capillaries into the urinary space are unknown. To answer this question, we developed an infection with Streptococcus pneumoniae (SP) in a humanized experimental IgAN model (α1KICD89tg mice) that resembles the pathological and clinical findings of disease (IgA1 and soluble CD89 mesangial deposits, complement activation, proteinuria and hematuria). Method α1KICD89tg mice (12 weeks old) received an intranasal instillation of SP (107 bacteria). Blood, urine and renal samples were obtained during 1 month after induction of respiratory infection. The presence of SP in lungs from these mice was confirmed by microbiological analysis. Hematuria was quantified in the urinary sediment and renal function was determined by biochemical analysis. Renal histological characteristics were evaluated by hematoxylin/eosin, masson's trichrome and PAS staining. IgA glomerular deposits, activation of complement system and infiltration of proinflammatory cells was examined by immunohistochemistry or immunofluorescence. Circulating leukocyte populations were studied on a hemocytometer. Renal inflammatory cytokines, metalloproteases, as well as markers of tubular and glomerular damage were determined in kidneys by RT-PCR and western-blot. To further validate the role of neutrophils in this pathological setting, we selective depleted these cells through a single injection of anti-Ly6G mAb (200 µg/kg i.p). Results SP-intranasal instillation in α1KICD89tg mice increased hematuria, microalbuminuria and proteinuria, peaking at 48h after induction of the respiratory infection. SP instillation caused disruption of the glomerular basement membrane, with decreased expression of the slit diaphragm proteins nephrin and synaptopodin, as well as higher glomerular accumulation of IgA and proteins of complement system (C3, MBL). Hematuria intensity was positively correlated with the presence of interstitial F4/80+ macrophages, matrix metalloproteinase 9 (MMP-9), inflammatory cytokines and chemokines (IL-1β, IL-6, TNF-α, CCL-2, CCL5 and CX3CL1/CX3CR1) as well as p65 NF-κB activation. Hematuria was negatively correlated with anti-inflammatory IL-10 mRNA expression, Factor H levels and collagen IV content. Notably, SP infection induced expression of the tubular injury markers N-GAL and KIM-1. Increased peripheral neutrophils levels were observed in the SP-infected α1KICD89tg mice. Mechanistically, anti-Ly6G-mediated neutrophil depletion reduced SP-mediated hematuria, proteinuria and albuminuria, prevented loss of synaptopodin and nephrin, decreased renal inflammation and MMP-9 expression in α1KICD89tg mice Conclusion In a humanized mouse model of IgAN, hematuria bouts following respiratory tract infections are caused by a neutrophil-mediated alteration of the glomerular filtration barrier (podocyte damage, complement deposits and loss of Collagen IV). These findings may help to unveil novel potential therapeutic approaches to combat one of the key elements in the progression of IgAN and related conditions.

Effects of Chemotherapy Agents on Circulating Leukocyte Populations: Potential Implications for the Success of CAR-T Cell Therapies.

Simple SummaryCAR-T cell therapy is a new approach to cancer treatment that is based on manipulating a patient’s own T cells such that they become able to seek and destroy cancer cells in a highly specific manner. This approach is showing remarkable efficacy in treating some types of blood cancers but so far has been much less effective against solid cancers. Here, we review the diverse effects of chemotherapy agents on circulating leukocyte populations and find that, despite some negative effects over the short term, chemotherapy can favourably modulate the immune systems of cancer patients over the longer term. Since blood is the starting material for CAR-T cell production, we propose that these effects could significantly influence the success of manufacturing, and anti-cancer activity, of CAR-T cells. Thus, if timed correctly, chemotherapy-induced changes to circulating immune cells could allow CAR-T cells to unleash more effective anti-tumour responses.Adoptive T-cell therapy using autologous T cells genetically modified to express cancer-specific chimeric antigen receptors (CAR) has emerged as a novel approach for cancer treatment. CAR-T cell therapy has been approved in several major jurisdictions for treating refractory or relapsed cases of B-cell precursor acute lymphoblastic leukaemia and diffuse large B-cell lymphoma. However, in solid cancer patients, several clinical studies of CAR-T cell therapy have demonstrated minimal therapeutic effects, thus encouraging interest in better integrating CAR-T cells with other treatments such as conventional cytotoxic chemotherapy. Increasing evidence shows that not only do chemotherapy drugs have tumoricidal effects, but also significantly modulate the immune system. Here, we discuss immunomodulatory effects of chemotherapy drugs on circulating leukocyte populations, including their ability to enhance cytotoxic effects and preserve the frequency of CD8+ T cells and to deplete immunosuppressive populations including regulatory T cells and myeloid-derived suppressor cells. By modulating the abundance and phenotype of leukocytes in the blood (the ‘raw material’ for CAR-T cell manufacturing), we propose that prior chemotherapy could facilitate production of the most effective CAR-T cell products. Further research is required to directly test this concept and identify strategies for the optimal integration of CAR-T cell therapies with cytotoxic chemotherapy for solid cancers.

Lipopolysaccharide endotoxin injections elevated salivary TNFα and corneal temperatures and induced dynamic changes in circulating leukocytes, inflammatory cytokines, and metabolic indicators in wether lambs.

Pathogenic infections increase morbidity and reduce performance in livestock, and thus understanding the comprehensive physiological changes associated with infections can benefit production sustainability. In this study, we sought to investigate such physiological responses to an acute immune challenge in lambs. Polypay wethers received single IV injections of 1.5 µg/kg lipopolysaccharide endotoxin (LPS-injected; n = 6) or saline (controls; n = 6). Corneal temperatures (via infrared thermography), rectal temperatures, blood, plasma, and saliva were assessed every 2 hr for 10 hr after injections. Blood was also assessed at 24 hr. LPS-injected lambs exhibited elevated (P < 0.05) corneal and rectal temperatures that peaked at 4 hr but were still slightly greater (P < 0.05) than controls at 10 hr. Circulating total white blood cells, monocytes, and granulocytes were reduced (P < 0.05) in LPS-injected lambs within the first 4 hr but were subsequently greater (P < 0.05) than in controls. Lymphocytes were reduced (P < 0.05) in LPS-injected lambs over the first 8 hr and did not differ from controls thereafter. Red blood cells, hematocrit, and hemoglobin were increased (P < 0.05) in LPS-injected lambs over the first 6 hr, indicating mild dehydration. Blood glucose briefly increased (P < 0.05) in LPS-injected lambs at 2 hr but was less (P < 0.05) than in controls thereafter. Blood lactate was greater (P < 0.05) in LPS-injected lambs between 6 and 10 hr after injections, which together with reduced (P < 0.05) CO2 partial pressure indicated a metabolic shift toward glycolysis. LPS-injected lambs exhibited a transient increase (P < 0.05) in plasma TNFα at 2 and 4 hr only and sustained increases (P < 0.05) in CXCL9 and CXCL10 beginning at 6 and 4 hr, respectively. They also exhibited a mild, paradoxical increase (P < 0.05) in the anti-inflammatory sFRP3. Salivary TNFα was increased (P < 0.05) in LPS-injected lambs at 2 hr only. Regression analyses indicated that rectal temperatures were a generally poor predictor of the other inflammatory components in this study, with the exception of circulating leukocyte populations. Likewise, correlations among the 10 cytokines measured in this study were generally weak, with notable exceptions between CXCL9 and CXCL10 and between IL-21 and IFNγ. These findings demonstrate that physiological changes to even short-lived immune challenges are dynamic in nature and persist beyond the time frame of febrile responses and other common assessments.

Stabilization of Human Whole Blood Samples for Multicenter and Retrospective Immunophenotyping Studies.

Whole blood is often collected for large-scale immune monitoring studies to track changes in cell frequencies and responses using flow (FC) or mass cytometry (MC). In order to preserve sample composition and phenotype, blood samples should be analyzed within 24 h after bleeding, restricting the recruitment, analysis protocols, as well as biobanking. Herein, we have evaluated two whole blood preservation protocols that allow rapid sample processing and long-term stability. Two fixation buffers were used, Phosphoflow Fix and Lyse (BD) and Proteomic Stabilizer (PROT) to fix and freeze whole blood samples for up to 6months. After analysis by an 8-plex panel by FC and a 26-plex panel by MC, manual gating of circulating leukocyte populations and cytokines was performed. Additionally, we tested the stability of a single sample over a 13-months period using 45 consecutive aliquots and a 34-plex panel by MC. We observed high correlation and low bias toward any cell population when comparing fresh and 6months frozen blood with FC and MC. This correlation was confirmed by hierarchical clustering. Low coefficients of variation (CV) across studied time points indicate good sample preservation for up to 6months. Cytokine detection stability was confirmed by low CVs, with some differences between fresh and fixed conditions. Thirteen months regular follow-up of PROT samples showed remarkable sample stability. Whole blood can be preserved for phenotyping and cytokine-response studies provided the careful selection of a compatible antibody panel. However, possible changes in cell morphology, differences in antibody affinity, and changes in cytokine-positive cell frequencies when compared to fresh blood should be considered. Our setting constitutes a valuable tool for multicentric and retrospective studies. © 2020 International Society for Advancement of Cytometry.

Analysis of the Progeny of Sibling Matings Reveals Regulatory Variation Impacting the Transcriptome of Immune Cells in Commercial Chickens.

There is increasing recognition that the underlying genetic variation contributing to complex traits influences transcriptional regulation and can be detected at a population level as expression quantitative trait loci. At the level of an individual, allelic variation in transcriptional regulation of individual genes can be detected by measuring allele-specific expression in RNAseq data. We reasoned that extreme variants in gene expression could be identified by analysis of inbred progeny with shared grandparents. Commercial chickens have been intensively selected for production traits. Selection is associated with large blocks of linkage disequilibrium with considerable potential for co-selection of closely linked “hitch-hiker alleles” affecting traits unrelated to the feature being selected, such as immune function, with potential impact on the productivity and welfare of the animals. To test this hypothesis that there is extreme allelic variation in immune-associated genes we sequenced a founder population of commercial broiler and layer birds. These birds clearly segregated genetically based upon breed type. Each genome contained numerous candidate null mutations, protein-coding variants predicted to be deleterious and extensive non-coding polymorphism. We mated selected broiler-layer pairs then generated cohorts of F2 birds by sibling mating of the F1 generation. Despite the predicted prevalence of deleterious coding variation in the genomic sequence of the founders, clear detrimental impacts of inbreeding on survival and post-hatch development were detected in only one F2 sibship of 15. There was no effect on circulating leukocyte populations in hatchlings. In selected F2 sibships we performed RNAseq analysis of the spleen and isolated bone marrow-derived macrophages (with and without lipopolysaccharide stimulation). The results confirm the predicted emergence of very large differences in expression of individual genes and sets of genes. Network analysis of the results identified clusters of co-expressed genes that vary between individuals and suggested the existence of trans-acting variation in the expression in macrophages of the interferon response factor family that distinguishes the parental broiler and layer birds and influences the global response to lipopolysaccharide. This study shows that the impact of inbreeding on immune cell gene expression can be substantial at the transcriptional level, and potentially opens a route to accelerate selection using specific alleles known to be associated with desirable expression levels.

Impact of systemic therapy on circulating leukocyte populations in patients with metastatic breast cancer

Tumors affect the immune system, locally and systemically. The frequencies of specific circulating immune cell populations correlate with disease progression as well as prognosis of the patients. Although largely neglected, conventional antitumoral therapies often possess immunomodulatory properties and affect the levels of specific immune cell populations. Most information, however, derive from animal or in vitro studies. As this could impact prognosis as well as response to therapy, further studies of the effects of treatment on circulating immune cells in patients are warranted. In this pilot study, we evaluated a wide panel of circulating immune cells over time (up to six months) in ten patients with metastatic breast cancer receiving standard antitumoral regimens. Overall, endocrine therapy tends to enrich for natural killer (NK) and natural killer T (NKT) cells in the circulation, whereas both chemotherapy and endocrine therapy reduce the levels of circulating monocytic myeloid-derived suppressor cells (Mo-MDSCs). This indicates that the systemic immunosuppressive profile observed in patients tends to revert over the course of systemic therapy and holds promise for future combination treatment with standard antitumoral agents and immunotherapy.

Expansion and activation of distinct central memory T lymphocyte subsets in complex regional pain syndrome

BackgroundComplex regional pain syndrome (CRPS) is a debilitating condition where trauma to a limb results in devastating persistent pain that is disproportionate to the initial injury. The pathophysiology of CRPS remains unknown; however, accumulating evidence suggests it is an immunoneurological disorder, especially in light of evidence of auto-antibodies in ~ 30% of patients. Despite this, a systematic assessment of all circulating leukocyte populations in CRPS has never been performed.MethodsWe characterised 14 participants as meeting the Budapest clinical criteria for CRPS and assessed their pain ratings and psychological state using a series of questionnaires. Next, we performed immunophenotyping on blood samples from the 14 CRPS participants as well as 14 healthy pain-free controls using mass cytometry. Using a panel of 38 phenotypic and activation markers, we characterised the numbers and intracellular activation status of all major leukocyte populations using manual gating strategies and unsupervised cluster analysis.ResultsWe have shown expansion and activation of several distinct populations of central memory T lymphocytes in CRPS. The number of central memory CD8+ T cells was increased 2.15-fold; furthermore, this cell group had increased phosphorylation of NFkB and STAT1 compared to controls. Regarding central memory CD4+ T lymphocytes, the number of Th1 and Treg cells was increased 4.98-fold and 2.18-fold respectively, with increased phosphorylation of NFkB in both populations. We also found decreased numbers of CD1c+ myeloid dendritic cells, although with increased p38 phosphorylation. These changes could indicate dendritic cell tissue trafficking, as well as their involvement in lymphocyte activation.ConclusionsThese findings represent the first mass cytometry immunophenotyping study in any chronic pain state and provide preliminary evidence of an antigen-mediated T lymphocyte response in CRPS. In particular, the presence of increased numbers of long-lived central memory CD4+ and CD8+ T lymphocytes with increased activation of pro-inflammatory signalling pathways may indicate ongoing inflammation and cellular damage in CRPS.

Flow-cytometric analysis of circulating leukocyte populations in turkeys: Establishment of a whole blood analysis approach and investigations on possible influencing factors

The blood cell counting methods used for diagnostic and research purposes in turkeys are, up to date, inferior to the techniques established for mammals and chickens. While microscopic counts are time consuming, previous flow cytometric approaches did not cover all blood cell types of interest due to the lack of turkey-specific markers for the different cell populations. Moreover, it is unknown to what extent the different leukocyte populations are affected by host and environmental factors including age, breed of the bird, and housing environment, respectively. In this study we established a whole blood based flow cytometric analysis method for turkeys. This method was used to determine baseline values depending on the age of the birds as well as under consideration of variations between trials and animal room effects. During three trials whole blood samples of B.U.T. 6 female turkeys were collected to analyze different leukocyte concentrations (cells/μl whole blood). In the first trial one group and in the second and third trial two groups with 22 birds/group were housed. Blood samples were collected at days one, 23, 43, 60, and 88 post hatch and concentrations of monocytes, MHC class II-positive, CD4+, and CD8+ lymphocytes, as well as thrombocytes and granulocytes were determined by flow cytometric analysis. Concentrations of all identified populations were not only influenced by the bird’s age (p < 0.05), they varied also among trials (p < 0.05) and even for some of the populations between animal rooms within the same trial despite comparable housing and management conditions. Therefore, for the establishment of baseline values for leucocyte concentrations in whole blood effects of age and housing have to be considered. In addition, our data emphasize the importance of the establishment of baseline values for different age groups, as age had the strongest effect on the blood cell numbers in this study.

Stress-induced mast cell activation contributes to atherosclerotic plaque destabilization

Mast cells accumulate in the perivascular tissue during atherosclerotic plaque progression and contribute to plaque destabilization. However, the specific triggers for mast cell activation in atherosclerosis remain unresolved. We hypothesized that psychological stress-induced activation of mast cells may contribute to plaque destabilization. To investigate this, apoE−/− mice on Western-type diet were exposed to 120′ restraint stress. A single episode of restraint caused a significant increase in mast cell activation in the heart. In addition to a rise in serum corticosterone and changes in circulating leukocyte populations, we observed an increase in the circulating pro-inflammatory cytokine interleukin (IL)-6 in the stressed mice. Subsequent characterization of the atherosclerotic plaques revealed a high incidence and larger size of intraplaque hemorrhages in stressed mice. In mast cell-deficient apoE−/− mice, restraint stress affected circulating leukocyte levels, but did not increase plasma IL-6 levels. Furthermore, we did not observe any intraplaque hemorrhages in these mice upon stress, strongly indicating the involvement of a mast cell-dependent response to stress in atherosclerotic plaque destabilization. In conclusion, we demonstrate that acute stress activates mast cells, which induces the incidence of intraplaque hemorrhage in vivo, identifying acute stress as a risk factor for atherosclerotic plaque destabilization.

Fingolimod reduces circulating tight-junction protein levels and in vitro peripheral blood mononuclear cells migration in multiple sclerosis patients

There are no data on the effects of fingolimod, an immunomodulatory drug used in treatment of multiple sclerosis (MS), on circulating tight-junction (TJ) protein levels as well as on peripheral blood mononuclear cells (PBMC) migration. Serum TJ protein [occludin (OCLN), claudin-5 (CLN-5) and zonula occludens-1 (ZO-1)] levels, sphingosine-1 phosphate 1 (S1P1) receptor expression on circulating leukocyte populations as well as in vitro PBMC migration were longitudinally assessed in 20 MS patients under 12-months fingolimod treatment and correlated with clinical and magnetic resonance imaging (MRI) parameters. After 12 months of treatment, a significant reduction of mean relapse rate as well as number of active lesions at MRI was found. TJ protein levels significantly decreased and were associated with reduction of S1P1 expression as well as of PBMC in vitro migratory activity. A significant correlation of CLN-5/OCLN ratio with new T2 MRI lesions and a significant inverse correlation of CLN-5/ZO-1 ratio with disability scores were found. These findings support possible in vivo effects of fingolimod on the blood-brain barrier (BBB) functional activity as well as on peripheral cell trafficking that could result in avoiding passage of circulating autoreactive cells into brain parenchyma. Circulating TJ protein levels and respective ratios could be further studied as a novel candidate biomarker of BBB functional status to be monitored in course of fingolimod as well as of other immunomodulatory treatments in MS.

Effect of imidacloprid ingestion on immune responses to porcine reproductive and respiratory syndrome virus

Nicotine and acetylcholine cause immunosuppresion by signaling to the α7 nicotinic acetylcholine receptor (α7 nAChR) on immune cells. Neonicotinoids are nAChR agonists and widly used insecticides. We aimed to define the immunosuppressive potential of dietary exposure to the neonicotinoid imidacloprid (IMI) on the generation of innate and adaptive immune responses to porcine reproductive and respiratory syndrome virus (PRRSV). Piglets were randomized into groups based on diet and infection. Behavioral signs of illness were recorded. Urine IMI levels were measured by high performance liquid chromatography-mass spectrometry. Flow cytometry was used to determine the expression pattern of the α7 nAChR on porcine leukocytes as well as the effects of infection and treatment on circulating leukocyte populations. Serum cytokines and PRRSV-specific antibody levels were determined by ELISA. Viral RNA in lung, spleen and plasma was determined by RT-qPCR. Pigs in the treatment group had elevated urine levels of IMI. Treatment with IMI reduced body weight, caused bouts of hypothermia, increased serum IL-10 and elevated levels of virus-specific antibodies. Viral RNA levels in the spleen showed a trend toward being increased in pigs fed IMI. Our data indicates that IMI injection may modulate virus specific immune function during PRRSV infection.