Phenotype Of Mononuclear Cells Research Articles

P-531. Mitochondrial DNA Haplogroups and Circulating Immune Cell Phenotypes in Veterans with and without HIV

Abstract Background HIV infection and mitochondrial function both influence cellular immune responses, but little is known about this relationship. We examined associations between mitochondrial DNA (mtDNA) haplogroups and peripheral blood mononuclear cell (PBMC) phenotypes among people with HIV (PWH) and without HIV in the Veterans Aging Cohort Study Biomarker Cohort (VACS-BC). Methods VACS-BC participants had mtDNA haplogroups determined from genome-wide genotyping and PBMC phenotyping by flow cytometry. Analyses included Kruskal-Wallis and Wilcoxon rank sum tests stratified by self-reported ancestry (non-Hispanic Black or White) and HIV status. Significant univariate associations were assessed in linear regression models adjusted for age and plasma viral load < 50 copies/mL among PWH. These exploratory analyses were not corrected for multiple comparisons. Results 1939 Veterans (1266 PWH; 95% male; median age 52 years [range 25-91]) had data available. In Black PWH (N=991), African haplogroup L3 was associated with lower % senescent (CD28-negative) CD4+ T cells (p=0.05), and haplogroup L2 was associated with higher % TEMRA CD8+ T-cells (p=0.03). In White PWH (N=275), European haplogroup T was associated with lower % CD38+ CD4+ T-cells (p=0.02) and haplogroup J with lower % IL4+ CD4+ T-cells (p=0.001). Haplogroups T and J were also associated with lower (p=0.05) and higher (p=0.02) % transitional memory CD8+ T-cells, respectively. Different age-adjusted associations were seen in Veterans without HIV, with the most prominent between haplogroups T and L1 and lower % total (CD14+) and classic (CD14+/CD16-) monocytes (p-value range=0.02-0.005). Conclusion MtDNA haplogroups were associated with cellular phenotypes, and associations differed by HIV status. Notable findings among PWH were in senescent, CD38+, and IL4-producing CD4+, and effector memory CD8+ T-cells, all important components of immune responses to viral infection. Haplogroup associations with monocyte populations in persons without HIV are intriguing and deserve further study. Limitations include those inherent in PBMC flow cytometry, cross sectional design, and small sample sizes for some subgroups. Validation in independent cohorts and mechanistic studies are needed. Disclosures Adeel A. Butt, MD, MS, Gilead Sciences: Grant/Research Support John R. Koethe, MD, Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|Merck & Co.: Advisor/Consultant|Merck & Co.: Grant/Research Support

Altered immune surveillance of B and T cells in patients with persistent residual lung abnormalities 12 months after severe COVID-19

BackgroundPost-COVID-19 respiratory sequelae often involve lung damage, which is called residual lung abnormalities, and potentially lead to chronic respiratory issues. The adaptive immune response, involving T-cells and B-cells, plays a critical role in pathogen control, inflammation, and tissue repair. However, the link between immune dysregulation and the development of residual lung abnormalities remains unclear.Methods109 patients discharged with residual lung abnormalities after a critical COVID-19 were followed for 12 months and divided as full recovery patients (FRG, n = 88) and persistent lung abnormalities (PLAG, n = 21). Cell profiling analysis was done using flow cytometry at 24 h of not antigen-specific in vitro stimulation. Plasma or supernatant levels of IFN-g, IL-4, IL-10, IgM, and IgG were assessed, and 10 patients (5 FRG, 5 PLAG) were randomly selected for detailed immune cell phenotyping and functional analysis of peripheral blood mononuclear cells using flow cytometry.ResultsCompared to the FRG group, PLAG exhibited an increase of unswitched (p = 0.0159) and decreased double-negative activated B-cells (p = 0.0317), systemic IL-10 levels were lower, displayed reduced frequency of total B-cells, and impaired spontaneous IgM (p = 0.0357) and IgG (p = 0.0079) release in culture. Regarding T-cells, PLAG patients showed a reduction in effector memory CD4 + cells (p = 0.0159) and an increase in CD4 + TEMRA cells (p = 0.0079) following in vitro stimulation. Notably, the PLAG group also exhibited higher frequencies of central memory CD4 + Th2 (GATA3+) T-cells in response to activation than the FRG group (p = 0.0079).ConclusionsPatients with residual lung abnormalities 12 months post-critical COVID-19 exhibit impaired B-cell function, increased unswitched B-cells, and higher frequencies of CD4 + TEMRA T-cells following in vitro activation. These immune imbalances may contribute to ongoing lung dysfunction and warrant further investigation as a potential mechanism in residual lung abnormalities. Larger studies are necessary to confirm these findings.

The ADIS study: Early Diagnosis of Alzheimer's Disease by Immune Profiling of Cytotoxic Lymphocytes and Recording of Sleep Disturbances

AbstractBackgroundSleep‐wake alterations are common symptoms in Alzheimer’s Disease (AD) associated with faster cognitive decline. Noradrenaline dysfunction and neuroinflammation have been proposed as potential driving mechanisms. The ADIS project aims to study the relationship between sleep‐wake patterns, immune signatures (peripheral blood cytotoxic lymphocytes), and noradrenergic markers across the AD spectrum.MethodThe project aims to recruit a total of 75 participants from the Hospital Clínic de Barcelona, including healthy controls (with normal values of plasma pTau), mild cognitive impairment due to AD (MCI), and AD Dementia (AD) confirmed by CSF biomarkers (Figure 1). All subjects will complete a comprehensive neuropsychological battery and Altoida digital assessment. Sleep‐wake phenotypes will be assessed by sleep questionnaires (Pittsburg Sleep Quality Questionnaire, PSQI, and Epworth Sleepiness Scale, ESS) and 2‐week actigraphy (Motion Wath 8 device). Noradrenaline levels and neuroinflammation markers in CSF will be measured. Immune phenotyping (cytometry, immune activation assays, scRNA/TCR sequencing) of peripheral blood mononuclear cells (PBMC) will be performed. In addition, selected participants will participate on the study's Advisory Board.ResultTo date, we have recruited 25 healthy controls, 25 MCI, and 23 AD participants who performed a total of 67 neuropsychological testings, 47 Altoida assessments, 34 CSF noradrenaline measurements, 65 actigraphy monitoring, and 59 isolation of PBMC. There were no age or gender differences between groups (Table 1). As expected, MMSE was lower in MCI/AD than controls (p<0.01). PSQI and ESS showed no differences, but preliminary analyses of 28 (10 HC, 14 MCI, 4 AD) actigraphy recordings showed that MCI and AD had a greater sleep latency than controls (p<0.05). As expected, preliminary analyses of 34 participants, including 21 MCI and 13 AD, showed similar noradrenaline levels. Further analyses will be performed, including the correlation between immune/noradrenaline markers and clinical data in an increased sample size.ConclusionSleep questionnaires might have a poor performance in identifying sleep alterations in MCI/AD individuals. Digitally assessed sleep‐wake patterns might help to detect sleep alterations. A deeper understanding of the role of biological mechanisms, such as immunologic profiles or noradrenergic dysfunction, driving sleep alteration is warranted.

Successful eculizumab treatment as an adjunctive therapy to desensitization in ABO-incompatible living donor kidney transplantation and its molecular phenotypes.

ABO-incompatible (ABOi) kidney transplantation (KT) has become an important option to overcome organ shortage. Plasmapheresis/rituximab-based desensitization therapy has successfully reduced anti-ABO antibody levels and suppressed antibody-mediated rejection (AMR) in ABOi KT. However, high titers of anti-ABO antibodies in some patients are refractory to standard desensitization, leading to loss of KT opportunities or AMR. Eculizumab treatment was used an adjunctive therapy to rescue high-titer ABOi KT patients refractory to plasmapheresis/rituximab-based desensitization. Molecular phenotypes of allograft biopsies and cellular phenotypes of peripheral blood mononuclear cells of eculizumab group were compared with those of control groups using the Banff Human Organ Transplant gene panel and flow-cytometric analysis, respectively. The initial titers of anti-ABO antibodies in the two patients were 1:512 and >1:1024; the final pre-transplant titers after desensitization were 1:128 and 1:64. Both patients received eculizumab from KT day to two or four weeks post-KT and maintained stable renal function up to one-year post-transplantation without overt infection, despite early episodes of probable AMR or borderline T cell-mediated rejection. Molecular phenotype analysis revealed that gene expression patterns in the ABOi KT with eculizumab group overlapped with those in the ABOi KT with AMR group more than in the ABOi KT without AMR group, except for complement pathway-related gene expression. Anti-ABO antibody titers decreased to low levels 1-3 months post-transplant in the eculizumab group in parallel with decreasing anti-B-specific B cells. Short-term eculizumab therapy is promising for rescuing ABOi KT recipients with high anti-ABO antibody titers refractory to plasmapheresis-based desensitization therapy.

Bone Marrow and Peripheral Blood Mononuclear Cell Phenotype Changes after Cultivation and Autologous Infusion in Patients with Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC) is a condition affecting the liver and immune system. In this study, the impact of autologous bone marrow-derived mononuclear cell (BM-MNC) transplantation on PBC patients was investigated. Sixteen eligible PBC patients participated at the National Scientific Medical Center in Astana, Kazakhstan, between 2017 and 2022, and BM-MNCs were harvested from their anterior iliac crest. After isolating and cultivating the BM-MNCs, they were infused back into the patient's peripheral veins. Changes in BM-MNC and peripheral blood mononuclear cell (PB-MNC) phenotypes were assessed before and after a 24-hour cultivation period and 72 hours post-transplantation. We monitored liver function parameters over 6-month intervals and conducted flow cytometry analysis to assess CD markers on BM-MNCs before and after cultivation and PB-MNCs before and after transplantation. Statistical analysis included the Friedman test for liver parameters and the Wilcoxon signed-rank test for BM-MNC and PB-MNC comparisons. Our findings revealed significant reductions in liver function tests after multiple transplantations. Flow cytometry analysis before and after a 24-hour culture and autologous BM-MNC infusion revealed the expansion of specific cell populations, with significant increases in CD3+, CD4+, CD16+, CD20+, CD25+, CD34+, CD105+, CD73+, СD117+, and CD34+populations, while CD4+25+, CD34+105+, and CD4+FOXP3+ populations decreased. Interestingly, a contradictory finding was observed with a decrease in bone marrow CD34+105+ cell lines (P=0.03) alongside an increase in peripheral CD34+105+ population (P=0.03). In summary, our study shows that BM-MNC transplantation in PBC patients leads to changes in immune cell populations and liver function. These findings suggest potential therapeutic applications of BM-MNC transplantation in managing PBC and offer insights into the dynamics of immune cells associated with this treatment approach.

TRLS-11. PNOC005: ONCOLYTIC MEASLES VIRUS FOR THE TREATMENT OF CHILDREN AND YOUNG ADULTS WITH RECURRENT MEDULLOBLASTOMA OR ATYPICAL TERATOID RHABDOID TUMOR

Abstract BACKGROUND PNOC005 is a phase 1 clinical trial investigating the safety and tolerability of intratumoral or intrathecal administration of oncolytic measles virus (MV-NIS) in children and young adults with recurrent medulloblastoma (MB) or atypical teratoid/rhabdoid tumor (ATRT). METHODS Patients with recurrence of MB or ATRT were stratified into Stratum A (local recurrence) or B (disseminated recurrence). Stratum A patients received MV-NIS directly into the tumor bed at time of surgical resection. Stratum B patients received a single dose of MV-NIS via lumbar puncture. After safety was demonstrated in Stratum A and B, Stratum C was added with repeat dosing on Day 0 and 7 for patients with recurrent disseminated MB. Specimens for viral shedding were collected. Blood was collected on days 0, 4, 7, 14, and 28. RNA-sequencing deconvolution and time-series analysis were performed to estimate the composition and phenotypes of peripheral blood mononuclear cells (PBMCs). RESULTS Thirty-four patients (median age 8.5, range 2-31) enrolled between February 2017 and April 2021, with 23 evaluable patients with MB and 4 with ATRT. One patient experienced a dose-limiting toxicity (grade 3 alanine aminotransferase increase). There were four MV-NIS-related adverse events grade 3 or greater across all strata. Viral shedding was detected in 5 patients, all of which cleared by end-of-treatment. We found a similar gene-expression program in PMBCs that correlated across patients (n=18), which was upregulated at days 4-7 post treatment and consistent with an antiviral response. Concomitant changes in B, T, and natural-killer cell composition and activation were consistent with this interpretation. CONCLUSION This is the first trial investigating intratumoral as well as repeated intrathecal delivery of MV-NIS in children with MB and ATRT. We show that therapy is safe and well-tolerated with minimal adverse effects. Immune markers and biologic correlates preliminarily indicate anti-viral effects in tumors.

New automation-friendly 96-well plate ready-to-use 8-color dry antibody panel for basic phenotyping of human whole blood and peripheral blood mononuclear cells using flow cytometry

Abstract Automation-friendly 96-well plate flow cytometry solutions are ideal for pharmaceutical, academic, and CRO laboratories that perform testing on large numbers of samples and/or large number of test conditions. Our ready-to-use pre-formulated dry antibody panel in 96-well plate format provide researchers all the reagents within the wells of the plate where the test is conducted in a stable dry format, resulting in dependable reagent performance and reduction of manual pipetting steps with the added benefit of time and material (reagent and sample) savings. In addition, the 96-well plate format is compatible with automated liquid handlers for walk-away flexibility and higher-throughput sample processing. Here we show flow cytometry data for both whole blood and PBMC samples stained with the first ready-to-use DURAClone IM Phenotyping BASIC plate (RUO) in a shallow 96-well plate containing a dry reagent antibody panel to identify major lymphocyte and monocyte subpopulations for the following markers: CD45, CD16, CD56, CD19, CD14, CD3, CD4 and CD8. Processing of whole blood samples using the shallow 96-well plate format was achieved using FLEXLyse, a lysing solution with low volume ratio of lysis/whole blood, applicable for no-wash and wash protocols. Our experiments were performed using automation on the Biomek i7 liquid handling automated workstation integrated with a CytoFLEX LX flow cytometer and microplate centrifuge demonstrating a fully automated flow cytometry workflow.

Mass cytometry reveals atypical immune profile notably impaired maturation of memory CD4 T with Gb3-related CD27 expression in CD4 T cells in Fabry disease.

Fabry disease (FD) is an X-linked disease characterized by an accumulation of glycosphingolipids, notably of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3) leading to renal failure, cardiomyopathy, and cerebral strokes. Inflammatory processes are involved in the pathophysiology. We investigated the immunological phenotype of peripheral blood mononuclear cells in Fabry patients depending on the clinical phenotype, treatment, Gb3, and lysoGb3 levels and the presence of anti-drug antibodies (ADA). Leucocytes from 41 male patients and 20 controls were analyzed with mass cytometry using both unsupervised and supervised algorithms. FD patients had an increased expression of CD27 and CD28 in memory CD45- and CD45 + CCR7-CD4 T cells (respectively p < 0.014 and p < 0.02). Percentage of CD45RA-CCR7-CD27 + CD28+ cells in CD4 T cells was correlated with plasma lysoGb3 (r = 0.60; p = 0.0036) and phenotype (p < 0.003). The correlation between Gb3 and CD27 in CD4 T cells almost reached significance (r = 0.33; p = 0.058). There was no immune profile associated with the presence of ADA. Treatment with agalsidase beta was associated with an increased proportion of Natural Killer cells. These findings provide valuable insights for understanding FD, linking Gb3 accumulation to inflammation, and proposing new prognostic biomarkers.

Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.

The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased the staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is a highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity.

Impacts of cryopreservation on phenotype and functionality of mononuclear cells in peripheral blood and ascites.

Mononuclear cells in peripheral blood and ascites are important clinical resources commonly used in translational and basic research. However, the impact of different cryopreservation durations and extra freeze-thaw cycles on the number and function of mononuclear cells is unknown. Peripheral blood samples (n = 21) and ascites samples (n = 8) were collected from healthy volunteers and ovarian cancer patients. Mononuclear cells were isolated, frozen, and thawed at 6 and 12 months. The impact of cryopreservation on cell viability, the phenotype, and the activation and proliferation of T cells were analyzed by flow cytometry. Single-cell sequencing was applied to investigate the underlying mechanism. The cell number and viability of mononuclear cells in peripheral blood and ascites were significantly decreased after cryopreservation. The T lymphocytes, especially CD4+ T cells, were affected the most significantly. By contrast, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, and B cells were more tolerant. Meanwhile, T cell proliferation and IL-2 secretion are significantly affected after long-term cryopreservation. Mechanistically, the cell death induced by elevated reactive oxygen species (ROS) was involved in the reduction of CD4+ T cells after cryopreservation. Our data indicates that different subtypes of mononuclear cells exhibit different tolerance capacities upon cryopreservation. Thus, our research can provide evidence and support for individuals who are conducting experiments using frozen clinical patient-derived mononuclear cells, for basic research or clinical trials. In addition, extra caution is worthwhile when researchers compare immune cell functionality from peripheral blood or ascites across datasets obtained in different cryopreservation conditions.

P1223 Lactobacillus rhamnosus GG/p40 encapsuled cationic host defense peptide play protective roles on immune-related colitis by inducing differentiation and immune suppression of regulatory T cells

Abstract Background The immune-related colitis (IRC) is the most prominent in immune-related adverse events (irAE). The association between gut microbiota and IRC development has been revealed. Our study aimed to explore the protective effect and mechanism of the probiotic Lactobacillus rhamnosus GG (LGG) and its secretory protein p40 on IRC. The self-assembled cationic host defense peptide (CHDP) hydrogel was applied to protect oral p40 protein from digestive damage and target enriched the site of colitis. Methods The IRC model mice were intervened with LGG by gavage (i.g.), p40 by enema (en.), or p40 encapsuled by CHDP (i.g.). The T cell phenotype of colonic lamina propria mononuclear cells (LPMC) was measured by flow cytometry. The indocyanine green (ICG) encapsuled by CHDP was gavage in IRC model mice, the distribution of CHDP was monitored by fluorescence signals imaging via IVIS, to assess the adhesive effect of CHDP in mice colitis. In vitro, the proportion of regulatory T cells (Treg) differentiated was measured by LGG supernatant co-cultured with CD4+ naïve T cells, and the division index of CD4+T cells was measured by LGG supernatant co-cultured with CD4+T cells and Treg (CD4+T cells: Treg=1:1) by carboxyfluorescein succinimidyl ester (CFSE) staining test. Results Compared with DSS+anti-PD-1+anti-CTLA-4 IRC model group, LGG (i.g.) group had lower weight loss, DAI score, histopathological score, relative IL-6 and TNF-α mRNA level, IL-6 and TNF-α concentration and higher colon length (P&amp;lt;0.05) (Fig.1A-L). P40 (en.) group had lower weight loss, DAI score, histopathological score and higher colon length (P&amp;lt;0.05) (Fig.1M-T). CHDP+p40 (i.g.) group had lower weight loss, DAI score, histopathological score, relative IL-6 and TNF-α mRNA level, IL-6 concentration and higher colon length (P&amp;lt;0.05) (Fig.1M-W). The percentages of CD25+FoxP3+Treg/CD4+T cells of colonic LPMC in LGG, p40 (en.), and CHDP+p40 (i.g.) group were significantly higher than IRC model group (P&amp;lt;0.05) (Fig.1X-Z, α). The average 28hr-residual rate of fluorescence imaging radiance in IRC-CHDP+ICG group was higher than both NC-CHDP+ICG and IRC-ICG group (Fig.2β-δ), and the average radiance of colon in IRC-CHDP+ICG group was also the highest (P&amp;lt;0.05) (Fig.2ε-ζ). In vitro, LGG 107 CFU/mL, 108 CFU/mL and 5 × 108 CFU/mL (P&amp;lt;0.05) could significantly promote Treg differentiation with concentration dependent manner (Fig.2η-ι), and the suppression of Treg on CD4+T cells with lower division index compared with control group (Fig.2κ). Conclusion LGG/p40 played protective role by promoting the differentiation and immune suppression of Treg cells in IRC. CHDP hydrogel could protect p40 protein from the digestive damage, and target enriched the inflamed lesion to improve the efficacy.

Accelarated immune ageing is associated with COVID-19 disease severity

BackgroundThe striking increase in COVID-19 severity in older adults provides a clear example of immunesenescence, the age-related remodelling of the immune system. To better characterise the association between convalescent immunesenescence and acute disease severity, we determined the immune phenotype of COVID-19 survivors and non-infected controls.ResultsWe performed detailed immune phenotyping of peripheral blood mononuclear cells isolated from 103 COVID-19 survivors 3–5 months post recovery who were classified as having had severe (n = 56; age 53.12 ± 11.30 years), moderate (n = 32; age 52.28 ± 11.43 years) or mild (n = 15; age 49.67 ± 7.30 years) disease and compared with age and sex-matched healthy adults (n = 59; age 50.49 ± 10.68 years). We assessed a broad range of immune cell phenotypes to generate a composite score, IMM-AGE, to determine the degree of immune senescence. We found increased immunesenescence features in severe COVID-19 survivors compared to controls including: a reduced frequency and number of naïve CD4 and CD8 T cells (p < 0.0001); increased frequency of EMRA CD4 (p < 0.003) and CD8 T cells (p < 0.001); a higher frequency (p < 0.0001) and absolute numbers (p < 0.001) of CD28−ve CD57+ve senescent CD4 and CD8 T cells; higher frequency (p < 0.003) and absolute numbers (p < 0.02) of PD-1 expressing exhausted CD8 T cells; a two-fold increase in Th17 polarisation (p < 0.0001); higher frequency of memory B cells (p < 0.001) and increased frequency (p < 0.0001) and numbers (p < 0.001) of CD57+ve senescent NK cells. As a result, the IMM-AGE score was significantly higher in severe COVID-19 survivors than in controls (p < 0.001). Few differences were seen for those with moderate disease and none for mild disease. Regression analysis revealed the only pre-existing variable influencing the IMM-AGE score was South Asian ethnicity (β\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\beta$$\\end{document} = 0.174, p = 0.043), with a major influence being disease severity (β\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\beta$$\\end{document} = 0.188, p = 0.01). ConclusionsOur analyses reveal a state of enhanced immune ageing in survivors of severe COVID-19 and suggest this could be related to SARS-Cov-2 infection. Our data support the rationale for trials of anti-immune ageing interventions for improving clinical outcomes in these patients with severe disease.

Egg-sensitised infants have elevated CD4+ effector memory T regulatory cells from birth.

IgE-mediated sensitisation to egg is common in infants. In some cases, the processes leading to egg sensitisation are established in early life, even before introduction to solid foods. The underlying mechanisms remain poorly understood. We performed detailed immune cell phenotyping of peripheral blood mononuclear cells and determined invitro cytokine responses following allergen specific and non-specific immune stimulation. To determine if unique immune profiles were linked to early-life egg sensitisation, we compared 92 infants at 4-6 months of age, with (EggCAP+, n = 41) and without (EggCAP-, n = 51) early egg sensitisation. Additionally, 47 cord blood samples were analysed. For a subset of participants (n = 39), matching cord blood mononuclear cells were assessed by flow cytometry to establish the impact of IgE sensitisation on immune developmental trajectories. EggCAP+ infants were found to exhibit a unique immune phenotype characterised by increased levels of circulating CD4+ T regulatory cells (Treg), CD4+ effector memory (EM) Treg and increased expression of the IgE receptor, FcεR1, on basophils. The increased CD4+ EM Treg profiles were already present in cord blood samples from EggCAP+ infants. A general Th2-skewing of the immune system was observed based on increased IL-13 production following phytohemagglutinin stimulation and by comparing immune developmental trajectories, EggCAP+ infants displayed an expansion of basophils and reduced levels of CD4- T cells compared to EggCAP- infants. Immunological profiles associated with egg sensitisation are detectable in infant circulation at 4-6 months of age and at birth. Understanding the immune mechanisms underlying early-life sensitisation could provide important insights for future food allergy prevention strategies.

Glycoprotein non-metastatic melanoma protein B expression correlates with the prognosis of acute liver injury/failure.

Background: Glycoprotein non-metastatic melanoma protein B (GPNMB) is expressed in macrophages during recovery from acute liver injury (ALI) in carbon tetrachloride (CCl4)-induced liver injury model mice. In this retrospective study, we assessed whether GPNMB levels in the serum and injured liver correlate with liver injury severity and prognosis in patients with ALI or acute liver failure (ALF). Methods: The study involved 56 patients with ALI or ALF who visited the Kagoshima University Hospital. Serum GPNMB level was measured over time, and the localization, proportion, origin, and phenotype of GPNMB-expressing cells in the injured liver were assessed. Finally, the phenotypes of human monocyte-derived macrophages and peripheral blood mononuclear cells (PBMCs) of patients with ALI and ALF were analyzed. Results: Peak GPNMB levels were significantly higher in patients with ALF and hepatic encephalopathy (HE), as well as in those who underwent liver transplantation or died, than in others. The peak GPNMB level correlated with prothrombin activity, prothrombin time-international normalized ratio, Model for End-stage Liver Disease score, and serum hepatocyte growth factor level. GPNMB was expressed in CD68-positive macrophages, and its level increased with the severity of liver injury. The macrophages showed the same polarization as M2c macrophages induced with interleukin-10 from human monocytes. Moreover, PBMCs from patients with ALF exhibited an immunosuppressive phenotype. Conclusion: We found that GPNMB levels in the serum and injured liver, which increased in patients with ALF, especially in those with HE, correlated with the severity of liver injury and prognosis of ALI and ALF. Moreover, GPNMB-positive macrophages exhibited the M2c phenotype. Our results indicate that persistently high GPNMB levels may be a prognostic marker in patients with ALI and ALF.

Role of Female Sex Hormones and Immune Response in Salt-Sensitive Hypertension Development: Evidence from Experimental Models.

Female sex hormones have systemic effects unrelated to their reproductive function. We describe experiences of different research groups and our own, on aspects related to the importance of female sex hormones on blood pressure (BP) regulation and salt-sensitivity-mediated BP response and salt sensitivity without alterations in BP, as well as renal sodium handling and interactions with the immune system. Changes in sodium intake in normotensive premenopausal women cause more BP variations than in men. After menopause, women often develop arterial hypertension (HT) with a profile of sodium sensitivity. Besides, experimental results have shown that in adult rat models resembling the postmenopausal hormonal state induced by ovariectomy, controlling BP is not enough to avoid renal and other tissue infiltration with immune cells, which does not occur when sodium intake is low or normal. Therefore, excess sodium promotes an inflammatory state with the involvement of immune cells. The evidence of activation of adaptive immunity, besides changes in T cell subpopulations, includes changes in sodium transporters and receptors. More studies are needed to evaluate the particular sodium sensitivity of women and its meaning. Changes in lifestyle and sodium intake reduction are the main therapeutic steps. However, to face the actual burden of salt-sensitive HT in postmenopausal women and its associated inflammatory/immune changes, it seems reasonable to work on immune cell activity by considering the peripheral blood mononuclear cell phenotypes of molecules and transport proteins related to sodium handle, both to screen for and treat cell activation.

Imbalance of Innate and Adaptive Immunity in Esophageal Achalasia.

Previous studies reveal that immune-mediated neuroinflammation plays a key role in the etiology of esophageal achalasia. However, the understanding of leucocyte phenotype and proportion is limited. This study aim to evaluate the phenotypes of leukocytes and peripheral blood mononuclear cells transcriptomes in esophageal achalasia. We performed high-dimensional flow cytometry to identified subsets of peripheral leukocytes, and further validated in lower esophageal sphincter histologically. RNA sequencing was applied to investigate the transcriptional changes in peripheral blood mononuclear cells of patients with achalasia. Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) was used for estimating the immune cell types. A differential gene expression analysis was performed and the differential expressed genes were subjected to gene ontology, Kyoto Encyclopedia of Genes and Genomes network, protein-protein interaction network construction. An imbalance between innate and adaptive immune cells occurred in achalasia. Specifically, neutrophils and CD8+ T cells increased both in peripheral blood and lower esophageal sphincter in achalasia. Eosinophils decreased in peripheral blood but massively infiltrated in lower esophageal sphincter. CIBERSORT analysis of peripheral blood mononuclear cells RNA sequencing displayed an increased prevalence of CD8+ T cells. 170 dysregulated genes were identified in achalasia, which were enriched in immune cells migration, immune response, etc. Proton pump inhibitor analysis revealed the intersections and gained 7 hub genes in achalasia, which were IL-6, Toll-like receptor 2, IL-1β, tumor necrosis factor, complement C3, and complement C1q A chain. Patients with achalasia exhibited an imbalance of systematic innate and adaptive immunity, which may play an important role in the development of achalasia.

Association of Juvenile Dermatomyositis Disease Activity With the Expansion of Blood Memory B and T Cell Subsets Lacking Follicular Markers.

This study was undertaken to identify blood markers of juvenile dermatomyositis (DM) disease activity (DA), which are needed to improve disease management. The study comprised a total of 123 juvenile DM patients and 53 healthy controls. Results of laboratory tests (aldolase, creatinine kinase, lactate dehydrogenase [LDH], aspartate aminotransferase) and clinical measures of DA in patients with juvenile DM, including the Manual Muscle Testing in 8 muscles (MMT-8), Childhood Myositis Assessment Scale (CMAS), and disease activity scores (DAS) (total DAS for juvenile DM, the muscle DAS, and the skin DAS), were recorded when available. Surface phenotype of peripheral blood mononuclear cells was assessed using flow cytometry. Whole blood transcriptional profiles were studied using either RNA-sequencing or microarrays. Differential gene expression was determined using DESeq and compared by pathway and gene ontology analyses. Conventional memory (CD27+IgD-) B cells expressing low CXCR5 levels (CXCR5low/- CM B cells) were significantly increased in frequency and absolute numbers in 2 independent cohorts of juvenile DM patients compared with healthy controls. The frequency of CD4+ Th2 memory cells (CD45RA-CXCR5-CCR6-CXCR3-) was also increased in juvenile DM, especially in patients who were within <1 year from diagnosis. The frequency of CXCR5low/- CM B cells correlated with serum aldolase levels and with a blood interferon-stimulated gene transcriptional signature. Furthermore, both the frequency and absolute numbers of CXCR5low/- CM B cells correlated with clinical and laboratory measures of muscle DA (MMT-8, CMAS, aldolase, and LDH). These findings suggest that both CM B cells lacking the CXCR5 follicular marker and CXCR5- Th2 cells represent potential biomarkers of DA in juvenile DM and may contribute to its pathogenesis.

A single, oral dose of the TLR7 agonist JNJ-64794964 induces transcriptomic and phenotypic changes in peripheral immune cells in healthy adults.

Chronic hepatitis B (CHB) is responsible for major disease burden worldwide. However, the number of available therapies is limited; cure remains an elusive goal. JNJ-64794964 (JNJ-4964) is an oral toll-like receptor-7 (TLR7) agonist being evaluated for the treatment of CHB. Here, we investigated the capacity of JNJ-4964 to induce transcriptomic and immune cell changes in peripheral blood in healthy volunteers. Peripheral blood was collected in the JNJ-4964 first-in-human phase 1 trial at multiple time points to assess transcriptomics and changes in frequency and phenotype of peripheral-blood mononuclear cells. Correlation of changes to JNJ-4964 exposure (Cmax) and changes in cytokine levels (C-X-C motif chemokine ligand 10 [CXCL10] and interferon alpha [IFN-α]) were evaluated. Fifty-nine genes, mainly interferon-stimulated genes, were up-regulated between 6hours and 5days after JNJ-4964 administration. JNJ-4964 increased frequencies of CD69, CD134, CD137, and/or CD253-expressing natural killer (NK) cells, indicative of NK cell activation. These changes correlated with Cmax, increase of CXCL10, and induction of IFN-α and were observed at IFN-α levels that are associated with no/acceptable flu-like adverse events. JNJ-4964 administration resulted in increased frequencies of CD86-expressing B cells, indicative of B-cell activation. These changes were predominantly observed at high IFN-α levels, which are associated with flu-like adverse events. JNJ-4964 administration led to changes in transcriptional profiles and immune cell activation phenotype, particularly for NK cells and B cells. Together, these changes could represent a set of biomarkers for the characterization of the immune response in CHB patients receiving TLR7 agonists.

Variations in dynamic tumor-associated antigen-specific T cell responses correlate with HCC recurrence after thermal ablation.

Ablative therapy is a recommended treatment for hepatocellular carcinoma (HCC) not only for its effective eradication of tumors, but also for its induction of host immunity. However, the high 5-year recurrence rate after ablation underlines the poor understanding of the antitumor immunity response. Here, we investigated the effects of thermal ablation on antitumor immunity. We analyzed the dynamics of tumor-associated antigen (TAA)-specific immune responses and changes in peripheral blood mononuclear cell phenotype in patients with HCC before and after tumor ablation. We used the IFN-γ ELISPOT assay and immunophenotyping by flow cytometry to evaluate the effects of ablation on host immunity. The correlation between the T cell response and disease outcome was explored to uncover the efficacy of the immune response in inhibiting HCC recurrence. Different TAA-specific T cell responses were identified among patients before and after ablation. One week after ablation, there was an improved immune state, with a switch from the dominance of an AFP-specific T cell response to that of a SMNMS-specific T cell response, which was correlated with better survival. Furthermore, an improvement in immune status was accompanied by a lower level of PD1+ and Tim3+ T cells in CD8+ T cells. Although this functional state was not durable, there was a higher degree of AFP-specific T cell responses at 4-weeks post-ablation. Furthermore, T cells presented a more exhausted phenotype at 4-weeks post-ablation than at the 1-week timepoint. Ablation elicits a transient antitumor immune response in patients with HCC by changing the profile of the T cell response and the expression of immune checkpoint molecules, which correlated with longer recurrence-free survival of patients with HCC.

New findings on the immune response following corneal transplantation

AbstractWe performed a multi‐centre, cross‐sectional analysis of the current immunological status of human full thickness (FT) and posterior lamellar (PL) corneal transplant (CT) recipients and control subjects using multiple technology platforms. The primary goal of this study was to define pathways associated with adverse immune responses to corneal transplantation and to generate and compare comprehensive, pathway‐based profiles of immune activity in CT acute rejection (AR). The secondary goal was to identify a composite biomarker of CT AR. Clinical details, including a comprehensive clinical examination, and blood samples, enabling analysis of peripheral blood mononuclear cell (PBMC) DNA, RNA and phenotype, were collected from adults with clinically diagnosed AR of FL‐CT (n = 35) and PL‐CT (n = 21) along with Stable CT recipients (n = 177) and adults with non‐transplanted corneal disease (n = 40). For those with AR, additional samples were collected 3 months later. CT acute rejection was clinically diagnosed and defined as precipitates on the corneal graft but not on the peripheral recipient cornea, either scattered or in the form of a Khodadoust line along with an increase in central corneal thickness. Immune cell analysis was performed by whole‐genome microarrays (whole blood) and high dimensional multi‐colour flow cytometry (peripheral blood mononuclear cells). For both, no activation signature was identified within the B cell and T cell repertoire at the time of AR diagnosis. Nonetheless, in FT‐ but not PL‐CT recipients, AR was associated with differences in B cell maturity and regulatory CD4+ T‐cell frequency compared to stable allografts. Our results suggest that, in contrast to solid organ transplants, genetic or cellular assays of peripheral blood are unlikely to be clinically exploitable for prediction or diagnosis of AR. However, further investigation of circulating B cell and T cell subpopulations may provide insights into the regulation of anti‐donor immune response in human CT recipients with differing AR risk.