Peripheral Blood Leukocyte Subsets Research Articles

Effects of parity and early pregnancy on peripheral blood leukocytes in dairy cattle

Subfertility remains a major problem in the dairy industry. Only 35-40% of high-yielding dairy cows and 55-65% of nonlactating heifers become pregnant after their first service. The immune system plays a critical role in the establishment of pregnancy. However, it can also create challenges for embryo survival and contribute to reduced fertility. We conducted 2 separate experiments to characterize changes in subsets of peripheral blood leukocytes (PBL) and their phenotype over the estrous cycle and early pregnancy in heifers and cows. We used flow cytometry and RT-qPCR to assess protein and mRNA expression of molecules important for immune function. We observed that CD14+ monocytes and CD3+ T cells tended to be affected by pregnancy status in heifers, whereas CD8B+ lymphocytes and NCR1+ natural killer (NK) cells were affected during early pregnancy in cows. Changes in expression of immune function proteins appeared to be greater in heifers than cows. To compare the most striking differences between heifers and cows observed in the initial experiments, we conducted a third experiment where PBL sampled from heifers and cows were simultaneously collected and analyzed under the same experimental conditions. Our results indicate that, compared with heifers, cows had greater mRNA expression of proinflammatory cytokines (IFNG and IL6) and AHR protein along with greater percentage of MM20A+ neutrophils and myeloid cells expressing SIRPA, ITGAM and ITGAX. Moreover, animals that failed to become pregnant showed altered expression of anti-inflammatory molecules compared with cyclic and pregnant animals. Overall, these findings support the hypothesis that early pregnancy signaling alters the phenotype of immune cells in the peripheral blood and that there are differences in the peripheral immune response to pregnancy between cows and heifers. Because cows have lower conception rates than heifers, it is possible that a more proinflammatory immune status in peripheral blood may play a role in embryo loss.

Absolute counts of leukocyte subsets in peripheral blood in pregnancies complicated by preterm prelabour rupture of membranes

Background This study aimed to assess variations in the absolute counts of various leukocyte subsets in the peripheral blood of women with pregnancies affected by preterm prelabour rupture of membranes (PPROM), in relation to the presence of intra-amniotic inflammation (IAI). Methods The study included fifty-two women with singleton pregnancies experiencing PPROM. Absolute counts of different leukocyte subpopulations, such as granulocytes, monocytes, lymphocytes, T cells and their subsets, B cells and their subsets, and NK cells and their subsets, were measured in maternal peripheral blood samples using multicolour flow cytometry. IAI was identified by elevated concentrations of interleukin 6 (IL-6) in the amniotic fluid, which was collected through transabdominal amniocentesis. Results Women with IAI exhibited higher absolute counts of leukocytes (p = 0.003), granulocytes (p = 0.008), and monocytes (p = 0.009). However, the presence of IAI did not significantly affect the absolute counts of lymphocytes or their subpopulations. Conclusions The study found that IAI is associated with changes in the absolute counts of leukocytes from the innate immunity compartment in the peripheral blood of women with pregnancies complicated by PPROM. Conversely, it does not significantly alter the counts of cells from the adaptive immune system. The changes observed may reflect the natural, temporal, and localised characteristics of IAI.

Transcriptional network analysis of peripheral blood leukocyte subsets in multiple sclerosis identifies a pathogenic role for a cytotoxicity-associated gene network in myeloid cells.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system affecting predominantly adults. It is a complex disease associated with both environmental and genetic risk factors. Although over 230 risk single-nucleotide polymorphisms have been associated with MS, all are common human variants. The mechanisms by which they increase the risk of MS, however, remain elusive. We hypothesized that a complex genetic phenotype such as MS could be driven by coordinated expression of genes controlled by transcriptional regulatory networks. We, therefore, constructed a gene coexpression network from microarray expression analyses of five purified peripheral blood leukocyte subsets of 76 patients with relapsing remitting MS and 104 healthy controls. These analyses identified a major network (or module) of expressed genes associated with MS that play key roles in cell-mediated cytotoxicity which was downregulated in monocytes of patients with MS. Manipulation of the module gene expression was achieved in vitro through small interfering RNA gene knockdown of identified drivers. In a mouse model, network gene knockdown modulated the autoimmune inflammatory MS model disease-experimental autoimmune encephalomyelitis. This research implicates a cytotoxicity-associated gene network in myeloid cells in the pathogenesis of MS.

Accurate enumeration of leukocyte subsets in peripheral blood with a 15-color immunoprofiling panel on a Cytek ®spectral flow cytometer

Abstract A white blood cell (WBC) differential is routinely performed on automated hematology analyzers as a general health check to evaluate immune status of patients. The recent advances in high parameter flow cytometry, especially full spectral flow cytometry, enable the analysis of more comprehensive leukocyte subsets to meet the increased demands in clinical research. We designed a 15-color, single-tube panel to identify and enumerate all major subsets of leukocytes on a Cytek full spectral flow cytometer. Human peripheral blood collected in K2EDTA, Heparin, ACD and Cyto-chex blood tubes were stained with 15 cFluor ®conjugated mAbs with a Lyse No Wash method and analyzed on a Cytek spectral flow cytometer. The performance of this assay was evaluated side by side with BD Multitest™ 6-color TBNK assay on BD FACSCanto II system on 13 healthy donor samples. Assay precision was examined on 12 donor samples in triplicates on three consecutive days. This 15-color panel identifies more than 20 leukocyte subpopulations: including neutrophils, eosinophils, basophils, lymphocytes, hematopoietic stem cells, monocyte subsets, T cell subsets, B cells, NK cell subsets. The results are comparable to that from BD 6-Color TBNK assay with R 2=0.961, 0.986, 0.940, 0.954, and 0.930 for CD3+, CD4+, CD8+ T, B and NK cells respectively. The correlations for all subsets are statistically significant with P < 0.001. The coefficient of variations (CV) of intra assay precision for all cell populations of interest are less than 25%. Cytek’s single-tube 15-color pan leukocyte panel demonstrated an effective, high sensitivity flow cytometry approach that can be used for monitoring immune cell subsets in peripheral blood in discovery and translational research.

Establishment of 14-Color Panel for Determining Leukocyte Subsets in Human Peripheral Blood with Flow Cytometry

To establish a 14-color flow cytometry protocol for the examination of leukocyte subsets in human peripheral blood. We used cell membrane surface antibodies CD45, CD3, CD4, CD8, CD19, CD56, CD16, CD14, CD25, CD127, HLA-DR, CD123, CD11c and nucleus staining dye DAPI to establish a 14-color flow cytometry assay to determine the major cell subsets in human peripheral blood. We collected peripheral blood specimens from healthy volunteers to test for antibody titers and optimal photomultiplier tube (PMT) voltage, and to conduct single-color staining and fluorescence minus one control staining. After determining the test method and test conditions, the peripheral blood samples of 18 healthy volunteers were analyzed. According to the cell classification and staining index, optimal antibody mass concentrations selected were as follows: CD25 and CD127 at 8.0 μg/mL, CD45, CD3, CD14 and CD123 at 4.0 μg/mL, CD8, CD19, CD56, CD16, HLA-DR and CD11c at 2.0 μg/mL, CD4 at 1.0 μg/mL and DAPI at 0.1 μg/mL. The detection voltages for CD45, CD3, CD4, CD8, CD19, CD56, CD16, CD14, CD25, CD127, HLA-DR, CD123, CD11c and DAPI were 450 V, 410 V, 400 V, 550 V, 405 V, 500 V, 520 V, 550 V, 550 V, 400 V, 450 V, 400 V, 580 V, and 300 V, respectively. The appropriate fluorescence compensation was determined by single-color staining and fluorescence minus one controls. The 14-color flow cytometry panel was established to analyze the main subsets of leukocytes in human peripheral blood, and peripheral blood samples from 18 healthy adults were examined, obtaining the percentages of each subset of peripheral blood leukocytes and the immunophenotypes of the main subsets. We established a 14-color panel for determining leukocyte subsets in human peripheral blood by flow cytometry, which produced stable and reliable results and was easy to operate.

P-075: Myeloid derived suppressor cells are not elevated in monoclonal gammopathies

<h3>Background</h3> The immunologically tolerant microenvironment may support a development of malignant multiple myeloma (MM) from precancerous monoclonal gammopathy of undetermined significance (MGUS) and blocks the efficacy of immunotherapy in existing MM as well. Myeloid derived suppressor cells (MDSC) represent heterogeneous group of cells with immunosuppressive activity and capability of promotion of tumor growth in MM. Their increased number and negative effect was described in MM but usually not in other monoclonal gammopathies (MGs). <h3>Aim</h3> Identification and enumeration of MDSC subsets in different MG subjects and comparison of their number with healthy controls. <h3>Methods</h3> Whole peripheral blood of 30 newly diagnosed and untreated MGs (2 MGUS, 7 MM with AL amyloidosis and 21 MM) and 13 healthy controls was used. Incubation with combination of MoAbs CD16/CD15/HLA-DR/CD14/CD45/CD11b/CD33/CD66b was done. Samples were analysed on BD FACSCanto II (BD Biosciences) after NH4Cl lysis and reanalysed with Infinicyt SW (Cytognos). <h3>Results</h3> Whole leukocytes were distributed into subpopulations using progressive gating strategy allowing detection of MDSC derived from granulocytes (G-MDSC) and monocytes (M-MDSC) with elimination of dendritic cells (DCs) and other contaminating cells (basophils etc.). There were found no differences in M- and G-MDSC relative and absolute count when compared whole MG group and controls. Even selection of MG with ≥95 % clonal plasma cells (PCs) or MGs with presence of circulating PCs or MGs with >30 g/l monoclonal Id protein did not show statistically different values of both MDSC subsets in comparison with control samples. The only statistically different value was decreased number of DCs in MGs. <h3>Conclusion</h3> Although elevated levels of MDSC in MM were previously published, this study did not prove it probably due to the different gating procedure. However, it was able to clearly define all leukocyte subsets in peripheral blood, thus only higher numbers of dublets may affect the final count of MDSC. Interestingly, low density neutrophils and tumor associated neutrophils should not be easily distinguish from MDSC, so their better characterization is needed in the future. The isolation and verification of MDSC immunosuppressive potential should be the next step in their analyses. Supported by MH CZ - DRO (FNBr, 65269705).

Abstract OT-27-01: PALVEN: A phase 1b study of palbociclib, letrozole and venetoclax in estrogen receptor, BCL2-positive metastatic breast cancer

Abstract Background: CDK4/6 inhibitors are integral to the treatment of Estrogen Receptor (ER) positive metastatic breast cancer (MBC). Although they are potent inhibitors of proliferation, tumor cell death (by apoptosis) may be curtailed. Venetoclax, an inhibitor of the pro-survival protein BCL2, has shown promise in an early phase clinical trial in ER+ MBC1. Moreover, preclinical studies suggest that venetoclax could improve tumor response to endocrine therapy and a CDK4/6 inhibitor by triggering apoptosis, including in growth arrested/senescent cells2. PALVEN is a phase 1b study (NCT NCT03900884), aiming to combine venetoclax with letrozole and the CDK4/6 inhibitor palbociclib. Trial Design: Eligible patients will be treated with letrozole (2.5 mg), palbociclib (75-125 mg) and venetoclax (100-800 mg) using a 3+3 dose escalation study design, with a maximum of 6 patients per dose cohort. Both palbociclib and venetoclax will be administered on day 1-21 of a 28 day cycle. Dose limiting toxicity (DLT) will be evaluated in the first 4 weeks of treatment. Tumor assessment will be performed every 8 weeks for 24 weeks and then every 12 weeks until progression. The primary endpoint is to describe DLTs reported within the first 4 weeks of treatment and determine the maximum tolerated dose (MTD), in order to define a recommended phase 2 dose (RP2D). Secondary endpoints include type and worst grade adverse events per patient (CTCAE v5.0), tumor response (RECIST v1.1), clinical benefit rate (CBR), progression free and overall survival (PFS, OS) as well as patient reported outcomes. Exploratory endpoints include metabolic response (using FDG-PET), changes in circulating tumor DNA (ctDNA), peripheral blood leukocyte subsets, and tumor phenotype in paired and progression biopsies. Eligibility Women with ER+ (≥10% positively stained carcinoma cells) and BCL2+ (≥50% cells with at least moderate cytoplasmic staining; intensity 2-3 on a 0-3 scale), unresectable locally advanced or MBC are eligible. Patients must have measurable or evaluable disease as per RECIST v1.1 and ECOG performance score of 0-1. Participants must not have had &amp;gt;2 prior lines of treatment in the metastatic setting and no previous treatment with CDK4/6 inhibitor or venetoclax in the adjuvant or metastatic setting. Statistical methods This is a proof-of-concept, dose escalation study and any statistical analysis of responses will be exploratory. Analysis will be focused primarily on adverse events, particularly DLTs reported in the DLT observation period. All secondary endpoints will be analysed separately combining all dosing cohorts. The response rate and CBR will be estimated with 95% confidence intervals calculated using exact methods based on the binomial distribution. Time-to-event endpoints (PFS and OS) will be described using Kaplan-Meier methods to calculate the median survival. Response rate, CBR and time-to-event endpoints (PFS and OS) will also be described for patients treated in the 1st versus 2nd and 3rd line setting. Accrual Target accrual is 6-36, depending on the number of dose cohorts required to reach DLT. Recruitment is active at 2 sites in Australia. References 1 Lok, S.W., et al. (2019). Cancer Discov 9, 354-369. 2 Whittle, J.R., et al. (2020). Clin Cancer Res Advance online. Citation Format: Christine Muttiah, Avraham Travers, James R Whittle, Sarah-Jane Dawson, Belinda Yeo, Jane E Visvader, Catherine Oakman, Geoffrey J Lindeman. PALVEN: A phase 1b study of palbociclib, letrozole and venetoclax in estrogen receptor, BCL2-positive metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-27-01.

Molecular, clinicopathological, and immune correlates of LAG3 promoter DNA methylation in melanoma.

BackgroundThe co-receptor lymphocyte-activation gene-3 (LAG3, LAG-3, CD223) is a potential target for immune checkpoint inhibition immunotherapies. However, little is known about the biological and clinical significance of LAG3 DNA methylation in melanoma and its microenvironment.MethodsWe evaluated LAG3 promoter and gene body methylation in a cohort of N = 470 melanoma patients obtained from The Cancer Genome Atlas (TCGA cohort), an independent cohort of N = 120 patients from the University Hospital Bonn, and in subsets of peripheral blood leukocytes, melanocytes, and melanoma cell lines. We validated the association of LAG3 methylation with mRNA expression in vitro in the melanoma cell line A375 treated with the hypomethylating agent 5-azacytidine and stimulated with interferon-γ. Finally, we investigated correlations between LAG3 methylation and progression-free survival in patients treated with immune checkpoint blockade (ICB cohort, N = 118).FindingsDepending on the analysed locus (promoter, gene body) we found region-dependent significant LAG3 methylation differences between monocytes, B cells, CD8+ and CD4+ T cells, regulatory T cells, melanocytes, and melanoma cell lines. In tumor tissues, methylation correlated significantly with LAG3 mRNA expression, immune cell infiltrates (histopathologic lymphocyte score and RNA-Seq signatures of distinct immune infiltrates), and an interferon-γ signature. Finally, LAG3 methylation was associated with overall survival in the TCGA cohort and progression-free survival in the ICB cohort. We detected basal LAG3 mRNA expression in the melanoma cell A375 and an interferon-γ inducible expression after demethylation with 5-azacytidine.InterpretationOur study points towards an epigenetic regulation of LAG3 via promoter methylation and suggests a prognostic and predictive significance of LAG3 methylation in melanoma. Our results give insight in the tumor cell-intrinsic transcriptional regulation of LAG3 in melanoma. In perspective, our results might pave the way for investigating LAG3 methylation as a predictive biomarker for response to anti-LAG3 immune checkpoint blockage.FundingA full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Thyroid stimulating hormone β-subunit splice variant is expressed in all fractional subsets of bone marrow hematopoietic cells and peripheral blood leukocytes and is modulated during bacterial infection

Thyroid stimulating hormone (TSH), a hormone produced in the anterior pituitary, is used to regulate thyroid hormone secretion. It has been known for over three decades that TSH is made by the cells of the immune system; however, the functional role of immune system TSH is unclear. We previously demonstrated that an alternatively-spliced isoform of TSHβ, referred to as the TSHβ splice variant (TSHβv), is the primary form of TSHβ made by hematopoietic cells in mice and humans. Most studies have linked TSHβv expression to myeloid cells of the immune system; however, it has recently been demonstrated that plasma cells in patients with Hashimoto’s thyroiditis may be a source of immune system TSHβv. Here, we demonstrate that TSHβv is expressed in bone marrow precursors of lymphoid cells, monocytes, and granulocytes, as well as in mesenteric lymph node (MLN) cells. Plasma cells generated by in vitro culture with bacterial lipopolysaccharide (LPS), and MLN cells from mice infected with L. monocytogenes expressed TSHβv. There was an increase in the intensity of intracellular TSHβv expression in MLN cells following exposure to LPS, and in the proportion of TSHβv+ CD138+ MLN cells following L. monocytogenes infection. The number of TSHβv+ cells increased in MLN cells, particularly among CD138+ cells, following bacterial infection. This was confirmed by an increase in gene expression of BLIMP-1, the transcription factor for CD138, following infection. Levels of circulating thyroxine dropped significantly in mice 24 hrs post-infection. These findings suggest that immune system TSHβv may contribute to the host immune response during bacterial infection.

Evaluation of Peripheral Blood Leukocyte Subsets in Systemic Lupus Erythematosus (SLE) Enabled by a Microfluidic Cell Separator

Abstract Although granulocyte signatures have been associated with SLE severity, their role in SLE has been poorly understood due to the suboptimal methods for granulocyte recovery from peripheral blood. An automated microfluidic technology (Sorterra™ prototype) was used to isolate all leukocytes with minimal platelet (PLT) or red blood cell (RBC) contamination to assess the cell subsets. Leukocytes were isolated from peripheral blood collected from healthy donors (HD) and SLE patients (3 female, 3 male in each group) using Sorterra, RBC lysis, or Ficoll®. Viability and recovery of each leukocyte subset was evaluated by cell counter and flow cytometry. Sorterra recovered 92±4% of leukocytes from HD and SLE samples with negligible RBC/PLT/microparticle (MP) contamination, whereas Ficoll isolated 35±15% of leukocytes, losing nearly all the granulocytes and retaining a substantial level of RBC/PLT/MP. With RBC lysis, up to 65% of neutrophils and 41% of eosinophils were lost in the process. Sorterra recovered more monocytes (~80%) than Ficoll (p&amp;lt;0.05). The recovery of lymphocytes by Sorterra (~90%) was not different from Ficoll or RBC lysis. The recovery of T and B cell subsets, NK, and NKT cells, was also not different among the isolation methods. The viability of Sorterra isolated leukocytes was significantly higher than the Ficoll isolated cells. The Sorterra prototype was superior to RBC lysis and Ficoll in recovering total leukocytes with high viability and ease of use and provided a more consistent leukocyte isolate from HD and SLE patients than other methods. The ability to recover granulocytes in their native state is unmatched by existing technologies and enables future studies to better elucidate the role of granulocytes in SLE development.

ASA Suppresses PGE2 in Plasma and Melanocytic Nevi of Human Subjects at Increased Risk for Melanoma.

Potential anti-inflammatory and anticarcinogenic effects of aspirin (ASA) may be suitable for melanoma chemoprevention, but defining biomarkers in relevant target tissues is prerequisite to performing randomized controlled chemoprevention trials. We conducted open-label studies with ASA in 53 human subjects with melanocytic nevi at increased risk for melanoma. In a pilot study, 12 subjects received a single dose (325 mg) of ASA; metabolites salicylate, salicylurate, and gentisic acid were detected in plasma after 4–8 h, and prostaglandin E2 (PGE2) was suppressed in both plasma and nevi for up to 24 h. Subsequently, 41 subjects received either 325 or 81 mg ASA (nonrandomized) daily for one week. ASA metabolites were consistently detected in plasma and nevi, and PGE2 levels were significantly reduced in both plasma and nevi. Subchronic ASA dosing did not affect 5” adenosine monophosphate-activated protein kinase (AMPK) activation in nevi or leukocyte subsets in peripheral blood, although metabolomic and cytokine profiling of plasma revealed significant decreases in various (non-ASA-derived) metabolites and inflammatory cytokines. In summary, short courses of daily ASA reduce plasma and nevus PGE2 and some metabolites and cytokines in plasma of human subjects at increased risk for melanoma. PGE2 may be a useful biomarker in blood and nevi for prospective melanoma chemoprevention studies with ASA.

Short-Term High-NaCl Dietary Intake Changes Leukocyte Expression of VLA-4, LFA-1, and Mac-1 Integrins in Both Healthy Humans and Sprague-Dawley Rats: A Comparative Study.

This study is aimed at assessing the effects of a short-term high-salt (HS) diet on the peripheral blood leukocyte (PBL) activation status in healthy rats and young human individuals. Distribution of PBL subpopulations and surface expression of integrins were determined using flow cytometry in 36 men and women on a 7-day low-salt diet (<3.2 g salt/day) immediately followed by a 7-day HS diet (~14 g salt/day) or in Sprague-Dawley (SD) rats (n = 24) on a 0.4% NaCl diet (aLS group) or a 4% NaCl diet (aHS group) for 7 days. The aHS group presented with an increased frequency of granulocytes, while the frequency of lymphocytes was reduced. Although in humans HS diet reduced the expression of CD11b(act) integrin on lymphocytes, the frequency of CD11b(act)-bearing cells among all PBL subsets was increased. The aHS group of rats exhibited increased expression of total CD11b/c in granulocytes and CD3 lymphocytes. The expression of CD11a was significantly reduced in all PBL subsets from human subjects and increased in the aHS group. CD49d expression on all PBL subsets was significantly decreased in both humans and rats. In human subjects, we found reduced frequencies of intermediate monocytes accompanied by a reciprocal increase in classical monocytes. Present results suggest that a short-term HS diet can alter leukocytes' activation status and promote vascular low-grade inflammation.

Discrimination of Head and Neck Squamous Cell Carcinoma Patients and Healthy Adults by 10-Color Flow Cytometry: Development of a Score Based on Leukocyte Subsets.

Background: Leukocytes in peripheral blood (PB) are prognostic biomarkers in head and neck squamous cell carcinoma cancer patients (HNSCC-CPs), but differences between HNSCC-CPs and healthy adults (HAs) are insufficiently described. Methods: 10-color flow cytometry (FCM) was used for in-depth immunophenotyping of PB samples of 963 HAs and 101 therapy-naïve HNSCC-CPs. Absolute (AbsCC) and relative cell counts (RelCC) of leukocyte subsets were determined. A training cohort (TC) of 43 HNSCC-CPs and 43 HAs, propensity score (PS)-matched according to age, sex, alcohol, and smoking, was used to develop a score consecutively approved in a validation cohort (VC). Results: Differences in AbsCC were detected in leukocyte subsets (p < 0.001), but had low power in discriminating HNSCC-CPs and HAs. Consequently, RelCC of nine leukocyte subsets in the TC were used to calculate 36 ratios; receiver operating characteristic (ROC) curves defined optimum cut-off values. Binary classified data were combined in a score based on four ratios: monocytes-to-granulocytes (MGR), classical monocytes-to-monocytes (clMMR), monocytes-to-lymphocytes (MLR), and monocytes-to-T-lymphocytes (MTLR); ≥3 points accurately discriminate HNSCC-CPs and HAs in the PS-matched TC (p = 2.97 × 10−17), the VC (p = 4.404 × 10−178), and both combined (p = 7.74 × 10−199). Conclusions: RelCC of leukocyte subsets in PB of HNSCC-CPs differ significantly from those of HAs. A score based on MGR, clMMR, MLR, and MTLR allows for accurate discrimination.

Circulating endothelial cells as biomarker for cardiovascular diseases

BackgroundEndothelial dysfunction is involved in several cardiovascular diseases. Elevated levels of circulating endothelial cells (CECs) and low levels of endothelial progenitor cells (EPCs) have been described in different cardiovascular conditions, suggesting their potential use as diagnostic biomarkers for endothelial dysfunction. Compared to typical peripheral blood leukocyte subsets, CECs and EPCs occur at very low frequency. The reliable identification and characterization of CECs and EPCs is a prerequisite for their clinical use, however, a validated method to this purpose is still missing but a key for rare cell events. ObjectivesTo establish a validated flow cytometric procedure in order to quantify CECs and EPCs in human whole blood. MethodsIn the establishment phase, the assay sensitivity, robustness, and the sample storage conditions were optimized as prerequisite for clinical use. In a second phase, CECs and EPCs were analyzed in heart failure with preserved (HFpEF) and reduced (HFrEF) ejection fraction, in arterial hypertension (aHT), and in diabetic nephropathy (DN) in comparison to age‐matched healthy controls. ResultsThe quantification procedure for CECs and EPCs showed high sensitivity and reproducibility. CEC values resulted significantly increased in patients with DN and HFpEF in comparison to healthy controls. CEC quantification showed a diagnostic sensitivity of 90% and a sensitivity of 68.0%, 70.4%, and 66.7% for DN, HFpEF, and aHT, respectively. ConclusionA robust and precise assay to quantify CECs and EPCs in pre‐clinical and clinical studies has been established. CEC counts resulted to be a good diagnostic biomarker for DN and HFpEF.

Abstract CT066: Dendritic cell based cryoimmunotherapy associates with clinical variables and changes in T-cell receptor expression in a prospective phase I trial of metastatic castration resistant prostate cancer

Abstract Interim analysis data are presented for 13 patients with metastatic castration resistant prostate cancer treated with dendritic cell based cryoimmunotherapy (CryoIT) and at least three months follow-up (FU). In CryoIT autologous immature dendritic cells (iDCs) mature in cryoablated tumor tissue and engulf tumor associated antigens and thereafter migrate to lymph nodes to instruct systemic immune attacks on heterogenous cancer cells. Primary endpoint of the trial is to investigate safety of CryoIT while secondary aims include clinical, radiological and immunological responses to treatment. Adverse events (AEs) and blood analyses were registered at all visits and imaging performed before and three months post-treatment. Norwegian regulatory authorities have approved the study. CryoIT of the prostate was performed as a freeze-thaw process under anesthesia and autologous iDCs injected after last thawing cycle. Radiologic responses according to RECISTv1.1 could categorize participants into either Group 1; stable disease (n=5) or Group 2; progressive cancer (n=4). Three patients showed mixed response and one had no follow-up imaging. Hematologic and immunologic values were evaluated according to response group. To identify peripheral blood leukocyte subsets and circulating tumor cells (CTCs), ultradeep T-cell receptor (TCR) β-chain sequences of complementarity-determining region 3, we utilized flow / mass cytometry, Cellsearch System, and TCRSafe Technology, respectively. At inclusion median age was 69 (62-73) years, BMI 25.9 (25-35), ECOG score 0 (0-1) and median PSA 8 (5-11). Group 2 demonstrated higher PSA (p=0.03), median 3.9 (IQR 2.7-6.8) and 74.6 (IQR 47.4-99.0) for Group 1 and Group 2, respectively. In total, 13 possible iDC-related AEs were reported by 10 individuals, whereof one severe and 12 mild-to-moderate, with urinary retention in seven subjects. All but one resolved by use of indwelling urinary catheter for median 8 (1-40) days. Participants with high CTC numbers (&amp;gt;5) before therapy had higher rates of progression, moderate pre-treatment CTC counts (3-5) decreased and no patient acquired CTCs after therapy. Ultradeep TCR-sequencing showed more prevalent and higher expression (&amp;gt;5-fold) of new TCR clonotypes 2-6 weeks after treatment in Group 1. The treatment seems safe and well tolerated. Higher PSA indicates more advanced disease at baseline for Group 2. The data indicate immune responses to treatment with higher increases in TCR clonotypes in radiologic stable disease. Clinical trial ID: NCT02423928. Citation Format: Liv Cecilie V. Thomsen, Alfred Honorè, Bjarte Almås, Lars A. Reisæter, Jannicke Frugård, Einar K. Kristoffersen, Guro Melve, Torjan Haslerud, Jarle Rørvik, Martin Biermann, Svein Inge Helle, Gunnar Kvalheim, Waqas Azeem, Jan Roger Olsen, Ole Johan Halvorsen, Lars Akslen, Duke K. Bahn, Klaus Pantel, Sabine Riethdorf, Haakon Ragde, Bjørn Tore Gjertsen, Anne M. Øyan, Karl-Henning Kalland, Christian Beisland. Dendritic cell based cryoimmunotherapy associates with clinical variables and changes in T-cell receptor expression in a prospective phase I trial of metastatic castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT066.

Birth weight and postnatal microbial exposures predict the distribution of peripheral blood leukocyte subsets in young adults in the Philippines

The immune system not only provides protection against infectious disease but also contributes to the etiology of neoplastic, atopic, and cardiovascular and metabolic diseases. Prenatal and postnatal nutritional and microbial environments have lasting effects on multiple aspects of immunity, indicating that immune processes may play important roles in the developmental origins of disease. The objective of this study is to evaluate the association between birth weight and the distribution of leukocyte (white blood cell) subsets in peripheral blood in young adulthood. Postnatal microbial exposures were also considered as predictors of leukocyte distribution. Participants (n=486; mean age=20.9 years) were drawn from a prospective birth cohort study in the Philippines, and analyses focused on the following cell types: CD4 T lymphocytes, CD8 T lymphocytes, B lymphocytes, natural killer cells, monocytes, granulocytes. Higher birth weight was a strong predictor of higher proportion of CD4 T lymphocytes (B=0.12, s.e.=0.041, P=0.003), lower proportion of CD8 T lymphocytes (B=-0.874, s.e.=0.364, P=0.016), higher CD4:CD8 ratio (B=1.964, s.e.=0.658, P=0.003), and higher B lymphocytes (B=0.062, s.e.=0.031, P=0.047). Measures of microbial exposure in infancy were negatively associated with proportions of B lymphocytes and granulocytes, and lower CD4:CD8 ratio. Leukocytes are the key regulators and effectors of innate and specific immunity, but the origins of variation in the distribution of cell type across individuals are not known. Our findings point toward nutritional and microbial exposures in infancy as potentially important determinants of immune-phenotypes in adulthood, and they suggest that leukocyte distribution is a plausible mechanism through which developmental environments have lasting effects on disease risk in adulthood.

The Influence of Cytomegalovirus on Expression of HLA-G and its Ligand KIR2DL4 by Human Peripheral Blood Leucocyte Subsets.

HLA-G is a non-classical class I HLA antigen, normally expressed in high levels only on extravillous cytotrophoblast. It has immunosuppressive properties in pregnancy and has also been found to be upregulated on leucocytes in viral infection. In this study, proportions of all leucocyte subsets expressing HLA-G were found to be low in healthy subjects positive or negative for cytomegalovirus (CMV). Significantly greater proportions of CD4+ CD69+ and CD56+ T cells expressed HLA-G compared to other T cells. However, following stimulation with CMV antigens or intact CMV, proportions of CD4+, CD8+, CD69+ and CD56+ T cells, and also B cells expressing HLA-G, were significantly increased in CMV+ subjects. Despite some subjects having alleles of HLA-G associated with high levels of expression, no relationship was found between HLA-G genotype and expression levels. Purified B cells from CMV+ subjects stimulated in mixed culture with CMV antigens showed significantly increased HLA-G mRNA expression by real-time polymerase chain reaction. Serum levels of soluble HLA-G were similar in CMV- and CMV+ subjects but levels in culture supernatants were significantly higher in cells from CMV+ than from CMV- subjects stimulated with CMV antigens. The HLA-G ligand KIR2DL4 was mainly expressed on NK cells and CD56+ T cells with no differences between CMV+ and CMV- subjects. Following stimulation with IL-2, an increase in the proportion of CD56+ T cells positive for KIR2DL4 was found, together with a significant decrease in CD56dimCD16+ NK cells. The results show that CMV influences HLA-G expression in healthy subjects and may contribute to viral immune evasion.

Leukocyte and plasma activation profiles in chronically transfused patients with a history of allergic reactions.

Allergic transfusion reactions are drawbacks to the benefits of transfusion. Classically, allergic transfusion reactions depend on histamine release from mast cells or basophils, but other leukocyte subsets may also be important. Thus, we propose to better define the exact leukocyte subsets involved in allergic transfusion reactions. The overall objective of the current study was to compare the activation of specific peripheral blood leukocyte subsets (monocytes, neutrophils, eosinophils, and basophils) in a cohort of 13 patients who received chronic transfusions and had a history of allergic transfusion reactions compared with a control group of patients who received chronic transfusions and had no history of allergic transfusion reactions. Leukocyte subsets were analyzed by flow cytometry at baseline and after red blood cell transfusion, and cytokine levels in platelet-free plasma collected at the same time points were measured by Luminex assay. Flow cytometry and cytokine profiles before and after transfusion did not differ significantly between patients who did and did not have a history of allergic transfusion reactions (p > 0.05). However, post-transfusion samples from both groups showed a decrease in CD63 expression in basophils, monocytes, and eosinophils and a decrease in CD45 expression in all leukocyte subsets compared with pretransfusion samples. Interleukin 10 levels increased after transfusion in the group with a history of allergic transfusion reactions (p = 0.0469), and RANTES (regulated upon activation, normal T-cell expressed and secreted) was significantly decreased post-transfusion in all patients (p = 0.0122). None of the leukocyte subsets from patients who had a history of allergic transfusion reactions significantly increased in activation either before or after transfusion. All leukocyte subsets from patients who did and did not have a history of allergic transfusion reactions decreased in their activation profile upon transfusion challenge.

Leukocyte counts and lymphocyte subsets in relation to pregnancy and HIV infection in Malawian women.

ProblemWe investigated leukocyte and lymphocyte subsets in HIV‐infected or HIV‐uninfected, pregnant or non‐pregnant Malawian women to explore whether HIV infection and pregnancy may act synergistically to impair cellular immunity.Method of studyWe recruited 54 pregnant and 48 non‐pregnant HIV‐uninfected women and 24 pregnant and 20 non‐pregnant HIV‐infected Malawian women. We compared peripheral blood leukocyte and lymphocyte subsets between women in the four groups.ResultsParturient HIV‐infected and HIV‐uninfected women had more neutrophils (each P<.0001), but fewer lymphocytes (P<.0001; P=.0014) than non‐pregnant women. Both groups had fewer total T cells (P<.0001; P=.002) and CD8+ T cells (P<.0001; P=.014) than non‐pregnant women. HIV‐uninfected parturient women had fewer CD4+ and γδ T cells, B and NK cells (each P<.0001) than non‐pregnant women. Lymphocyte subset percentages were not affected by pregnancy.ConclusionMalawian women at parturition have an increased total white cell count due to neutrophilia and an HIV‐unrelated pan‐lymphopenia.

Experimental acute infection of alpacas with Bovine viral diarrhea virus 1 subgenotype b alters peripheral blood and GALT leukocyte subsets.

Bovine viral diarrhea virus (BVDV) is a pathogen in cattle and alpacas ( Vicugna pacos), causing acute and persistent BVDV infections. We characterized the effect of acute BVDV infection on the immune system of alpacas by determining lymphocyte subpopulations in peripheral blood and gut-associated lymphoid tissues (GALT) as well as serum interferon levels. Alpacas were experimentally infected with BVDV-1b (strain CO-06). Peripheral blood leukocytes were isolated at 0, 3, 6, and 9 d postinfection (dpi), and leukocytes of GALT at 9 dpi, and evaluated using flow cytometry. Serum interferon levels were determined daily. Flow cytometric analyses of peripheral blood leukocytes showed a significant decrease in CD4+, CD8+, and αβ T-lymphocytes at 3 dpi. CD8+ lymphocytes were significantly increased, and activated lymphocytes were significantly decreased in the C3-stomach region in BVDV-infected alpacas. Serum interferon concentrations significantly increased in BVDV-infected alpacas at 3-6 dpi, peaking at 3 dpi. Our study confirms that BVDV can be a primary acute pathogen in alpacas and that it induces an interferon response and alters leukocyte subset populations. The changes in the proportion of T-lymphocytes during the early stages of BVDV infection may result in transient immunosuppression that may contribute to secondary bacterial and viral infections, similar to cattle.