Peripheral Blood Mononuclear Cell Populations Research Articles

102 Combination and individual vaccines for bovine viral diarrhea virus and infectious bovine rhinotracheitis effects on reproductive cyclicity and immune response

Abstract Vaccines are known to impact both the immune and reproductive systems. The objective of this research was to identify immunological and cyclic differences following vaccination for Bovine Viral Diarrhea Virus (BVDV) and/or Infectious Bovine Rhinotracheitis (IBR). Brahman cows (n = 15) were administered PGF2α to regress corpus lutea on d -3. Animals (d 0) were immunized (2mL, intramuscular injection) with one of three treatments: 1) Modified Live Vaccine (MLV) for BVDV and IBR (BVD+IBR; n = 5), 2) MLV for BVDV only (BVD; n = 5), or 3) sterile saline (CON; n = 5). Blood samples (30 mL/cow) were collected (d -3, 0, 2, 4, 6, 8, 10, and 14) and peripheral blood mononuclear cells (PBMC) were isolated. PBMC were incubated with propidium iodide and antibodies for specific surface cell markers (CD4, CD8, CD25, CD14, CD86, and CD335). An Amnis FlowSight flow cytometer determined cell type percentage. BVDV and IBR antibody concentrations were analyzed in serum samples (d -3, 0, and 14). Plasma samples (d 0, 2, 4, 6, 8, 10, and 14) were evaluated for interferon (IFN)-γ, interleukin (IL)-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17A, macrophage inflammatory protein (MIP)-1α, MIP-1β, IL-36RA, interferon-gamma-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF) α, and vascular endothelial growth factor (VEGF) A concentrations by a MagPix multiplex machine using a MILLIPLEX Bovine Cytokine/Chemokine Magnetic Bead Panel. Progesterone (d 0, 2, 4, 6, 8, 10, and 14) and estradiol (d 0) concentrations were determined by RIA. Differences in antibody titers, PBMC populations, and cytokine concentrations were analyzed as repeated measures using PROC MIXED (SASv9.4). Forty percent of BVD+IBR animals experienced abnormal estrus cycles, but no BVD or CON animals did. There was a treatment effect on BVD titer concentration with BVD animals having greater titer concentrations than CON (P = 0.02) but similar concentrations to BVD+IBR (P = 0.18). There was a treatment by time interaction (P < 0.0001) on IBR titers with BVD+IBR animals having greater titer concentrations on d 14 compared with BVD and CON. Treatment had no effect on leukocyte populations (P ≥ 0.1409), but all populations differed over time (P ≤ 0.0045). Antigen presenting cells (CD14+) and natural killer cells (CD335+) were affected by a treatment and time interaction (P ≤ 0.0479). BVD animals had increased CD14+ cells on d 2, 4, 8, and 14 compared with CON, but CON had a greater CD335+ cell population compared with BVD and BVD+IBR on d 10. There tended to be or was a significant treatment by day interaction for IL-1α (P = 0.08), IL-1b (P = 0.03), IL-10 (P = 0.06), IFN-γ (P = 0.05), IP-10 (P = 0.02), MIP-1α (P = 0.02), MIP-1 b (P = 0.01), TNFα (P = 0.01), and VEGFA (P = 0.03). IL-1a, IL-10, IL-1b, and MIP-1 b increased in BVD+IBR from d 0 to 4 or 6 then decreased but not in CON or BVD. Increased VEGFA was observed in CON compared with BVD and BVD+IBR animals. Overall, BVD+IBR resulted in abnormal cycles and changes in cytokine concentrations and leukocyte populations. USDA is an equal opportunity provider and employer.

Immune cell activity during anti-TNF treatment in patients with psoriasis and psoriatic arthritis.

Psoriasis is a chronic, inflammatory skin disease characterized by a dysregulated immune response and systemic inflammation. Up to one-third of patients with psoriasis have psoriatic arthritis (PsA). Targeted treatment with antibodies neutralizing tumor necrosis factor (TNF) can ameliorate both diseases. We here explored the impact of long-term infliximab treatment on the composition and activity status of circulating immune cells involved in chronic skin and joint inflammation. Immune cells were analyzed by multicolor flow cytometry. We measured markers of immune activation in peripheral blood mononuclear cell (PBMC) populations in 24 infliximab-treated patients with psoriasis/psoriatic arthritis compared to 32 healthy controls. We observed a significant decrease in the frequency of both peripheral natural killer (NK) cells and their subset CD56dimCD16+ NK cells in PsA compared to healthy controls and patients with psoriasis. The latter had a strong positive correlation with PASI in these patients, while CD56brightCD16- NK cells were negatively correlated with PASI. In addition, we observed an upregulation of CD69+ intermediate CD14+CD16+ and CD69+ classical CD14+CD16- monocytes in PsA and increased activity of CD38+ intermediate CD14+CD16+ monocytes in patients with psoriasis. Compared to healthy controls, psoriasis patients demonstrated shifts of the three B cell subsets with a decrease in transitional CD27-CD38high B cells. Our exploratory study indicates a preserved pathophysiological process including continuous systemic inflammation despite clinical stability of the patients treated with infliximab.

Association of CD24, CD27, and co-stimulatory molecules CD80 immunological marker expression on B-cells of human peripheral blood with development of celiac disease

Abstract: BACKGROUND: B-cells express a variety of clusters of differentiation markers during development. These markers determine the basic function of the immune phenotype specific to B-cells. Changes in the expression of these markers are linked to the development of many diseases, including chronic inflammation, autoimmune diseases, and immunodeficiency. OBJECTIVE: The current study aimed to investigate a change in CD24, CD27, and co-stimulatory molecules CD80 expression on peripheral blood B-cells and the extent of their contribution to celiac disease. MATERIALS AND METHODS: A total of 60 male children, whose ages ranged between 8 and 14 years, participated in this study. Thirty-five were identified as having celiac disease, while the control group comprised 25 children with anti-tissue transglutaminase (TG) (immunoglobulin G [IgG]), anti-tissue TG (IgA), and deamidated gliadin peptide (DGP) IgG levels normal. The frequency CD24, CD27, and CD80 expression were measured by flow cytometry. RESULTS: Celiac disease patients showed a substantial decrease in the percentage of CD24, CD27, and CD80 expression on B-cells compared to control groups. CONCLUSION: These findings suggest that numerical deficiency of CD24, CD27, and CD80 expression on B-cells in the peripheral blood mononuclear cell population, that may involve the loss of auto-tolerance that plays an important role in the immune response associated with inflammation and tissue damage in celiac disease. These immunological markers may be used as diagnostic indicators for this disease.

Abstract PO4-09-09: Deep Immunoprofiling Demonstrates Racial Differences in the Peripheral Immune System in Women at Risk for Breast Cancer

Abstract Background: Disparities in breast cancer outcomes between Non-Hispanic Black (NHB) and Non-Hispanic White (NHW) women have been attributed to NHB women presenting with more aggressive subtypes and later stage disease, differences in treatment receipt and adherence, access to care and tumor biology. However, previous work has shown that these known factors do not fully account for the observed disparities in outcomes. Systemic immune fitness is a factor not previously studied that may play a role in dictating disease progression and therapeutic responses. Numerous factors, including age and race, impact immune system function and understanding these changes is critical to address disparities in breast cancer outcomes. Here, we developed a pipeline for deep immunoprofiling of peripheral blood mononuclear cells (PBMC) at single cell resolution by full-spectrum flow cytometry. Using this pipeline, we sought to investigate whether there are differences in immune fitness between NHB and NHW women at high risk for developing breast cancer. Methods: PBMC were collected and processed from age-matched NHB and NHW women (n=20/cohort) identified as being at high risk for developing breast cancer through our institution’s B-PREP (Breast Cancer Personalized Risk Assessment Education and Prevention) clinic. Two custom antibody panels and protocols for comprehensive immunoprofiling of T and B cell subsets (T/B Panel), as well as monocytic, NK, and dendritic cell subsets (M/N/D Panel), along with functional and exhaustion markers were designed, optimized, and implemented. Traditional gating strategies were used to quantify proportions of canonical cell populations, and unbiased high dimensionality reduction analysis was performed using the OMIQ analysis platform to assess phenotypes and functional status of various immune cell subsets. Results: Significant differences between cohorts in abundance as well as phenotype of various cell populations were observed. Specifically, CD4+ follicular helper T cells, TEMRA CD8, and CD45RA Terminal Effector CD8+ cells were enriched in NHB women while PD-1+/CD3+ cells and PD-1+/CD8+ cells were enriched in the NHW women (Table). Additionally, several immunosuppressive and systemic inflammatory immune cell types were enriched in NHB women (Table). Conclusions: Deep immunoprofiling using single-cell spectral flow cytometry revealed differences in peripheral immune cells between NHB and NHW women at high risk for breast cancer. The significant enrichment of immunosuppressive monocytes and several activated NK cell populations in PBMC samples from NHB women relative to those from NHW women indicate an altered immune environment that may influence breast cancer development. Additional analysis, including a larger sample size and patients with invasive cancer, are ongoing to further understand if differences in immune fitness between NHB and NHW women are associated with disparities in breast cancer outcomes. Table: PBMC populations (% of CD45+) that are significantly enriched in NHB relative to NHW patients Citation Format: Esther Ogayo, Milos Spasic, Tasnim Rahman, Olga Kantor, Tari King, Peter van Galen, Sandra McAllister, Elizabeth Mittendorf. Deep Immunoprofiling Demonstrates Racial Differences in the Peripheral Immune System in Women at Risk for Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-09-09.

Preclinical evaluation and first-in-dog clinical trials of PBMC-expanded natural killer cells for adoptive immunotherapy in dogs with cancer

BackgroundNatural killer (NK) cells are cytotoxic cells capable of recognizing heterogeneous cancer targets without prior sensitization, making them promising prospects for use in cellular immunotherapy. Companion dogs develop spontaneous cancers...

Imprinted immune abnormalities in liver transplant patients cured of hepatitis C with antiviral drugs.

Chronic HCV infection induces interferon and dysregulates immune responses through inflammation and chronic antigenic stimulation. Antiviral drugs can cure HCV, providing a unique opportunity to examine the immunological restoration that does and does not occur when a chronic viral infection is eradicated. We quantified blood cytokines levels and used mass cytometry to immunophenotype peripheral blood mononuclear cells before and after HCV cure in 2 groups of patients and controls. At baseline, serum interferon α and soluble CD163 (a macrophage product) were elevated in both liver transplant and nonliver transplant patients compared to controls; the frequencies of several peripheral blood mononuclear cell populations differed from controls; and programmed death protein 1-positivity was increased in nearly all T cell subsets. Many abnormalities persisted after HCV cure, including elevated programmed death protein 1 expression on CD4 naïve and central memory T cells, elevated soluble CD163, and expansion of the plasmablast/plasma cell compartment. Several myeloid-lineage subsets, including Ag-presenting dendritic cells, remained dysregulated. In mechanistic studies, interferon α treatment increased programmed death protein 1 on human T cells and increased T cell receptor signaling. The data identify immunological abnormalities that persist after curative HCV treatment. Before cure, high levels of interferon α may stimulate programmed death protein 1 expression on human T cells, causing persistent functional changes.

Systemic Neutrophil Gelatinase-Associated Lipocalin Alterations in Chronic Pancreatitis: A Multicenter, Cross-Sectional Study.

Chronic pancreatitis (CP) is a progressive fibroinflammatory disorder lacking therapies and biomarkers. Neutrophil gelatinase-associated lipocalin (NGAL) is a proinflammatory cytokine elevated during inflammation that binds fatty acids (FAs) such as linoleic acid. We hypothesized that systemic NGAL could serve as a biomarker for CP and, with FAs, provide insights into inflammatory and metabolic alterations. NGAL was measured by immunoassay, and FA composition was measured by gas chromatography in plasma (n = 171) from a multicenter study, including controls (n = 50), acute and recurrent acute pancreatitis (AP/RAP) (n = 71), and CP (n = 50). Peripheral blood mononuclear cells (PBMCs) from controls (n = 16), AP/RAP (n = 17), and CP (n = 15) were measured by cytometry by time-of-flight. Plasma NGAL was elevated in subjects with CP compared with controls (area under the curve [AUC] = 0.777) or AP/RAP (AUC = 0.754) in univariate and multivariate analyses with sex, age, body mass index, and smoking (control AUC = 0.874; AP/RAP AUC = 0.819). NGAL was elevated in CP and diabetes compared with CP without diabetes ( P < 0.001). NGAL + PBMC populations distinguished CP from controls (AUC = 0.950) or AP/RAP (AUC = 0.941). Linoleic acid was lower, whereas dihomo-γ-linolenic and adrenic acids were elevated in CP ( P < 0.05). Linoleic acid was elevated in CP with diabetes compared with CP subjects without diabetes ( P = 0.0471). Elevated plasma NGAL and differences in NGAL + PBMCs indicate an immune response shift that may serve as biomarkers of CP. The potential interaction of FAs and NGAL levels provide insights into the metabolic pathophysiology and improve diagnostic classification of CP.

Mononuclear cell composition and activation in blood and mucosal tissue of eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic, inflammatory, antigen-driven disease of the esophagus. Tissue EoE pathology has previously been extensively characterized by novel transcriptomics and proteomic platforms, however the majority of surface marker determination and screening has been performed in blood due to mucosal tissue size limitations. While eosinophils, CD4+ T cells, mast cells and natural killer (NK) T cells were previously investigated in the context of EoE, an accurate picture of the composition of peripheral blood mononuclear cells (PBMC) and their activation is missing. In this study, we aimed to comprehensively analyze the composition of peripheral blood mononuclear cells and their activation using surface marker measurements with multicolor flow cytometry simultaneously in both blood and mucosal tissue of patients with active EoE, inactive EoE, patients with gastroesophageal reflux disease (GERD) and controls. Moreover, we set out to validate our data in co-cultures of PBMC with human primary esophageal epithelial cells and in a novel inducible mouse model of eosinophilic esophagitis, characterized by extensive IL-33 secretion in the esophagus. Our results indicate that specific PBMC populations are enriched, and that they alter their surface expression of activation markers in mucosal tissue of active EoE. In particular, we observed upregulation of the immunomodulatory molecule CD38 on CD4+ T cells and on myeloid cells in biopsies of active EoE. Moreover, we observed significant upregulation of PD-1 on CD4+ and myeloid cells, which was even more prominent after corticosteroid treatment. With co-culture experiments we could demonstrate that direct cell contact is needed for PD-1 upregulation on CD4+ T cells. Finally, we validated our findings of PD-1 and CD38 upregulation in an inducible mouse model of EoE. Herein we show significant alterations in the PBMC activation profile of patients with active EoE in comparison to inactive EoE, GERD and controls, which could have potential implications for treatment. To our knowledge, this study is the first of its kind expanding the multi-color flow cytometry approach in different patient groups using in vitro and in vivo translational models.

IgG from patients with mild or severe COVID‑19 reduces the frequency and modulates the function of peripheral mucosal-associated invariant T cells in PBMCs from healthy individuals.

Lower levels of peripheral mucosal-associated invariant T (MAIT) cells have been observed in the peripheral blood of patients with severe coronavirus disease 2019 (COVID-19). Following on from previous research into the effect of the IgG repertoire on human lymphocytes, the present study aimed to evaluate if immunoglobulin G (IgG) antibodies obtained from patients with mild or severe COVID-19 contribute to these effects on MAIT cells. Culture experiments were performed using healthy human peripheral blood mononuclear cells (PBMCs) and different repertoires of IgG obtained from patients with COVID-19 as a mild or severe disease and compared with mock, healthy control or therapeutic IgG conditions. The results indicate that the IgG repertoire induced during the development of mild and severe COVID-19 has, per se, the in vitro potential to reduce the frequency of MAIT cells and the production of IFN-γ by the MAIT cell population in PBMCs from healthy individuals. In conclusion, the results of the present study indicate that IgG in patients with severe COVID-19 may participate in the reduction of peripheral MAIT cell frequency and hinder the antiviral activity of these cells.

MicroRNA Expression Levels in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus Type 1 Positive Individuals and Relationship with Different Levels of Viral Suppression.

The persistence of low human immunodeficiency virus type 1 (HIV-1) replication in individuals undergoing antiretroviral therapy (ART) still threatens their health. Previous findings have shown that microRNAs (miRNAs) could interfere with several steps of the viral life cycle. Herein, we set out to investigate the expression of miR-150, miR-223, miR-382, miR-324-5p, miR-33a-5p, miR-34a, and miR-132 in the whole peripheral blood mononuclear cell (PBMC) population from people living with HIV-1 showing different levels of viral suppression. Levels of PBMC-associated miRNAs were analyzed in 30 individuals with undetectable viremia (target not detected) and 30 individuals with detectable low-level viremia (1-200 copies/mL). In addition, 30 samples from treatment-naive (NAIVE) individuals were investigated. Results were compared to a control group of 28 HIV-negative donors. All miRNAs analyzed were strongly downregulated in the NAIVE population, either compared to the treated group or to controls. Stratification of ART-treated donors according to the therapeutic regimen showed the downregulation of miR-33a-5p in subjects treated with non-nucleoside reverse transcriptase inhibitors compared with those treated with protease inhibitors. Collectively, the present study shows that uncontrolled viral replication leads to profound miRNA deregulation while treated individuals, irrespective of the degree of viral suppression, and even the types of antiviral drugs seem to be specifically associated with miRNA expression profiles. These evidences suggest that virological suppression could be favored by miRNA modulation.

Immunogenicity, safety, and preliminary efficacy evaluation of OVX836, a nucleoprotein-based universal influenza A vaccine candidate: a randomised, double-blind, placebo-controlled, phase 2a trial

OVX836, a recombinant vaccine containing the nucleoprotein of the influenza A virus A/WSN/1933 (H1N1) and the oligomerisation domain OVX313, has displayed a good safety profile and elicited dose-dependent humoral and cellular immune responses at 90 μg or 180 μg (intramuscularly) in previous clinical trials. The aim of this study was to explore higher doses, since no maximum tolerated dose had been reached. In this phase 2a, randomised, double-blind, placebo-controlled study, we recruited 137 healthy adults aged 18-55 years in a single centre in Belgium. Participants were randomly assigned (interactive web response system; block size=4) using SAS (version 9.4) to receive one single intramuscular administration of OVX836 influenza vaccine at three doses (180 μg [n=33], 300 μg [n=35], and 480 μg [n=36]) or placebo (n=33). The two primary endpoints were the safety and the cell-mediated immune response to OVX836 at the three doses in terms of change of nucleoprotein-specific IFNγ spot forming cell (SFC) frequencies in the peripheral blood mononuclear cell (PBMC) population, measured by IFNγ ELISpot, at day 8 versus pre-injection baseline (day 1). The population used for the safety analysis is the modified intention-to-treat cohort. The population used for the immunogenicity analysis is the per-protocol cohort. This trial is registered with ClinicalTrials.gov, NCT05060887, and EudraCT, 2021-002535-39. Participants were recruited between Nov 15, 2021, and Feb 1, 2022. OVX836 had a favourable safety profile up to 480 μg without reaching the maximum tolerated dose, and showed a good safety profile at all doses with mild local and systemic reactogenicity. 7 days after vaccination, although no significant differences were observed between the doses, OVX836 increased the frequency of nucleoprotein-specific IFNγ SFCs per million PBMCs from days 1 to 8 (primary endpoint): by 124 SFCs per 106 PMBCs (95% CI 67 to 180; p=0·002) at 180 μg; by 202 SFCs per 106 PMBCs (95% CI 138 to 267; p<0·0001) at 300 μg; by 223 SFCs per 106 PMBCs (95% CI 147 to 299; p<0·0001) at 480 μg; and decreased by 1 SFCs per 106 PMBCs (95% CI -24 to 22] in the placebo group (Kruskal-Wallis test p<0·0001 followed by Mann-Whitney's tests; per-protocol cohort). Dose-dependent and polyfunctional nucleoprotein-specific CD4 T-cell responses were observed, and CD8 T-cell responses were elicited at 300 μg and 480 μg (secondary endpoints). OVX836 appears to be a safe and well tolerated candidate vaccine that elicits humoral and cellular nucleoprotein-specific immune responses (including CD8 T cells at the highest dose levels) and showed a preliminary signal of protection against influenza. Therefore, OVX836 is a promising vaccine candidate for universal influenza A prevention, that warrants further trials. OSIVAX, Bpifrance, Wallonia Region, and the EUs Horizon 2020 Research and Innovation Program.

201-OR: Characterization of Type 1 Diabetes (T1D) Endotypes

To better understand how T1D heterogeneity relates to disease progression and therapeutic responsiveness, we studied 566 recent onset subjects (mean age at onset: 16.5 +/- 8.2) that had participated in the following TrialNet trials: MMF-DZB, anti-CD20; GAD-alum; CTLA4-Ig; Canakinumab; and ATG-GCSF. Our analysis began with a bioassay where plasma collected pre-intervention and within 100 days of diagnosis was used to induce transcription in a well-controlled peripheral blood mononuclear cell population. Microarray analysis identified 2,854 transcripts exhibiting high variation in at least 5 out of 6 trials. Hierarchical clustering and principal component analysis of these transcripts concordantly divided the participants into 2 major groups: Group 1 and Group 2. Analysis of post-baseline samples found group assignments conserved over time. Proportional analyses of the 406 subjects aged &amp;lt;18 years found Groups 1 and 2 distinct: subjects aged &amp;gt;14years segregated within Group 2 (39.9% vs 29.3%, p=0.03); subjects possessing neutral/low-risk HLA haplotypes segregated within Group 1 (20.5% vs 11.8%, p=0.04); placebo arm participants with the most rapid C-peptide rate of decline (&amp;lt;-0.15) segregated within Group 1 (20.3% vs 7.1%, p=0.04); and the subjects most responsive to anti-CD20 (p=0.01) and ATG-GCSF (p=0.07) segregated within Group 1. A local validation cohort (n=66, mean age at onset: 11.1 +/- 4.0) was analyzed by plasma induced transcription. A Random Forest model, which utilized the TrialNet data as a training set, was used to assign the local subjects to Group 1 or 2 (ROC: AUC=0.95; p=7.7E-9). Consistent with pathway analysis of the TrialNet transcriptomic data, ELISA analysis identified elevated plasma IFNγ, TNFα, IL-1β, IL-6, IL-12p70 levels (p&amp;lt;0.05) in Group 1. Flow cytometry measured higher percentages CD4+ CD45RO+ memory T cells (among total CD4 T cells, p=0.01) in Group 1. These analyses support the existence of two major T1D endotypes that exhibit differing immune phenotypes and therapeutic responsiveness. Disclosure A.Bedrat: None. T.Pant: None. S.Jia: None. M.Roethle: None. N.A.Truchan: None. S.M.Cabrera: Research Support; Abbott Diabetes. Y.Chen: None. C.Lin: None. M.Hessner: None. Funding National Institutes of Health (R01DK121528)

Proteomic analysis of circulating immune cells identifies cellular phenotypes associated with COVID-19 severity

Certain serum proteins, including C-reactive protein (CRP) and D-dimer, have prognostic value in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nonetheless, these factors are non-specific, providing limited mechanistic insight into the peripheral blood mononuclear cell (PBMC) populations that drive the pathogenesis of severe COVID-19. To identify cellular phenotypes associated with disease, we performed a comprehensive, unbiased analysis of total and plasma-membrane PBMC proteomes from 40 unvaccinated individuals with SARS-CoV-2, spanning the whole disease spectrum. Combined with RNA sequencing (RNA-seq) and flow cytometry from the same donors, we define a comprehensive multi-omic profile for each severity level, revealing that immune-cell dysregulation progresses with increasing disease. The cell-surface proteins CEACAMs1, 6, and 8, CD177, CD63, and CD89 are strongly associated with severe COVID-19, corresponding to the emergence of atypical CD3+CD4+CEACAM1/6/8+CD177+CD63+CD89+ and CD16+CEACAM1/6/8+ mononuclear cells. Utilization of these markers may facilitate real-time patient assessment by flow cytometry and identify immune populations that could be targeted to ameliorate immunopathology.

The Enigma of Lymphocyte Apoptosis in the Response to Influenza Virus Infection

In the pathogenesis of influenza virus infection, lymphocyte apoptosis as a part of the infection and/or the immune response to the virus can be somewhat puzzling. The percentage of human T lymphocytes within the peripheral blood mononuclear cell population that becomes apoptotic greatly exceeds the percentage that are infected after exposure to the virus, consistent with substantial apoptosis of bystander T lymphocytes. Studies reveal an important role of viral neuraminidase expression by co-cultured monocyte/macrophages in induction of apoptosis, including that of uninfected bystander lymphocytes. Despite these observations, it is a reasonable perspective to recognize that the development of lymphocyte apoptosis during the response to infection does not preclude a successful immune response and recovery of the infected host in the great majority of cases. Further investigation is clearly warranted to understand its role in the pathogenesis of influenza virus infection for human subjects.

Influence of conditioned medium from squamous cell carcinoma of the tongue on lymphoblasts and peripheral blood mononuclear cells.

Squamous cell carcinoma (SCC) is the most common malignant neoplasm of the oral cavity and is associated with high morbidity and mortality. Attention has been given to the role of inflammatory cells in carcinogenesis because of the ability of cancer cells to subvert the immune response. However, little is known about how molecules from neoplastic cells interact with lymphoblasts and circulating immune cells. This study aimed to understand the mechanisms by which SCC cells modulate the immune response by analyzing the influence of conditioned medium derived from SCC cell lines on immune cells. Lymphoblastic cells (CEM) and peripheral blood mononuclear cells (PBMC) were cultured in a conditioned medium derived from squamous cell carcinoma cells (SCC9 or SCC4) and analyzed for cell viability, CD4/CD8/FOXP3 profile by flow cytometry, and chemokine levels. Conditioned medium derived from SCC4 and SCC9 presented higher concentrations of IL-6 and IL-8 than IL-1β, IL-10, and IFN-γ. CEM and PBMCs when cultured with conditioned medium derived from SCC4 and SCC9 reduced IL-1β, IL-8, and IFN-γ concentrations. Conditioned medium from SCC4 increased CD4+ population in both CEM and PBMCs, while in conditioned medium from SCC9 it occurred only in PBMCs. PBMCs when cultured with both conditioned mediums increased CD8+ /FOXP3+ cells. CEM cells when cultured with conditioned medium derived from SCC4 and SCC9 reduced. Collectively, our results suggest that the products derived from squamous cell carcinoma on inflammatory cells can promote an immunosuppressed environment by reducing cell viability, changing cytokine expression, and altering the cell immunoprofile.

Altered transcription factor targeting is associated with differential peripheral blood mononuclear cell proportions in sarcoidosis.

IntroductionIn sarcoidosis, peripheral lymphopenia and anergy have been associated with increased inflammation and maladaptive immune activity, likely promoting development of chronic and progressive disease. However, the molecular mechanisms that lead to reduced lymphocyte proportions, particularly CD4+ T-cells, have not been fully elucidated. We posit that paradoxical peripheral lymphopenia is characterized by a dysregulated transcriptomic network associated with cell function and fate that results from altered transcription factor targeting activity.MethodsMessenger RNA-sequencing (mRNA-seq) was performed on peripheral blood mononuclear cells (PBMCs) from ACCESS study subjects with sarcoidosis and matched controls and findings validated on a sarcoidosis case-control cohort and a sarcoidosis case series. Preserved PBMC transcriptomic networks between case-control cohorts were assessed to establish cellular associations with gene modules and define regulatory targeting involved in sarcoidosis immune dysregulation utilizing weighted gene co-expression network analysis and differential transcription factor involvement analysis. Network centrality measures identified master transcriptional regulators of subnetworks related to cell proliferation and death. Predictive models of differential PBMC proportions constructed from ACCESS target gene expression corroborated the relationship between aberrant transcription factor regulatory activity and imputed and clinical PBMC populations in the validation cohorts.ResultsWe identified two unique and preserved gene modules significantly associated with sarcoidosis immune dysregulation. Strikingly, increased expression of a monocyte-driven, and not a lymphocyte-driven, gene module related to innate immunity and cell death was the best predictor of peripheral CD4+ T-cell proportions. Within the gene network of this monocyte-driven module, TLE3 and CBX8 were determined to be master regulators of the cell death subnetwork. A core gene signature of differentially over-expressed target genes of TLE3 and CBX8 involved in cellular communication and immune response regulation accurately predicted imputed and clinical monocyte expansion and CD4+ T-cell depletion.ConclusionsAltered transcriptional regulation associated with aberrant gene expression of a monocyte-driven transcriptional network likely influences lymphocyte function and survival. Although further investigation is warranted, this indicates that crosstalk between hyperactive monocytes and lymphocytes may instigate peripheral lymphopenia and underlie sarcoidosis immune dysregulation and pathogenesis. Future therapies selectively targeting master regulators, or their targets, may mitigate dysregulated immune processes in sarcoidosis and disease progression.

Effect of Porcine Colostral Exosomes on T Cells in the Peripheral Blood of Suckling Piglets.

Simple SummaryExosomes in porcine colostrum have gained attention as the possible key compounds involved in the growth and/or development of suckling piglets. In this study, peripheral blood mononuclear cells (PBMCs) from suckling piglets were cultured with or without milk-derived exosomes (control) in vitro. Porcine colostral exosomes increased the proportion of cytotoxic T (Tc) cells, while this phenomenon was not observed in PBMC whose endocytosis was inhibited. Moreover, exosome-treated PBMCs had a higher cytokine IL-2 concentration in the culture supernatant than the control. The present study demonstrated that porcine colostral exosomes could increase the Tc cell proportion in the peripheral blood of a suckling piglet, with the underlying mechanism believed to be the stimulation of IL-2 production in PBMCs via endocytosis.Growing evidence indicates that porcine colostral exosomes may contribute to the healthy development of piglets. Here, we evaluated in vitro the effect of porcine milk-derived exosomes, in particular colostral exosomes, on T cells in the peripheral blood of suckling piglets. A total of seven sows and thirteen suckling piglets were used. Peripheral blood mononuclear cells (PBMCs) from suckling piglets were cultured with or without milk-derived exosomes (control). Using flow cytometry, the proportion of each T cell subset in cultured PBMCs was analyzed three days post-incubation. PBMCs cultured with porcine colostral exosomes had a higher proportion of CD3+CD4−CD8+ T cells (cytotoxic T cells; Tc) than the control. However, exosomes induced no increase in the Tc cell population in PBMC whose endocytosis was inhibited. We further measured the concentrations of cytokines in the culture supernatant. Exosome-treated PBMCs had a higher cytokine IL-2 concentration than the control. The present study demonstrated that porcine colostral exosomes could increase the Tc cell proportion in the peripheral blood of suckling piglets, with the underlying mechanism believed to be the stimulation of IL-2 production in PBMCs via endocytosis. Moreover, our results suggested that porcine colostral exosomes were involved in the development of cellular immunity in suckling piglets.

Increased expressions of CD123, CD63, CD203c, and Fc epsilon receptor I on blood leukocytes of allergic asthma.

Background : Altered basophil identification markers have been discovered to associate with allergic asthma (AA) in recent years. However, little is known about the expression of basophil markers in blood granulocytes. Aim: To parallel test blood basophils in peripheral blood mononuclear cell (PBMC) and granulocyte populations of patients with AA and AA combined with allergic rhinitis (ARA) Methods: The expressions of surface molecules were determined via flow cytometry. CD123 expressing cells in blood were isolated using a cell sorting technique, and mouse AA models were employed for in vivo study. Results: The numbers of CD123+HLA-DR− cells in the granulocytes of AA and ARA patients markedly increased. However, only 49.7% of CD123+HLA-DR− cells in granulocytes and 99.0% of CD123+HLA-DR− cells in PBMCs were basophils. Almost all CD123+HLA-DR− cells expressed CD63 regardless in granulocytes or PBMC. The numbers of CD63, Fc epsilon receptor I (FcεRI), and CD203c expressing cells markedly enhanced in CD123+HLA-DR− granulocytes of AA and ARA patients. Mean fluorescence intensity (MFI) of CD63 and CD203c expressions on CD123+HLA-DR− PBMC and granulocytes of AA and ARA patients dramatically elevated. House dust mite extract (HDME) and Artemisia sieversiana wild allergen extract (ASWE) enhanced the numbers of CD63+CD123+HLA-DR− granulocytes and PBMC and the MFI of CD203c expression on CD123+HLA-DR− granulocyte of AA and ARA patients. Histamine, tryptase, and PGD2 enhanced proportions of CD123+ KU812 cells. ASWE- and HDME-induced AA mice showed upregulated CD63 expression on basophils. In conclusion, upregulated expressions of CD123, CD203c, CD63, and FcεRIα in PBMC and granulocytes of patients with AA and ARA suggest that CD123+HLA-DR− cells may contribute to the development of AA and ARA.

The Neonatal Fc Receptor Is Elevated in Monocyte-Derived Immune Cells in Pancreatic Cancer.

The neonatal Fc receptor (FcRn) is responsible for recycling of IgG antibodies and albumin throughout the body. This mechanism has been exploited for pharmaceutic delivery across an array of diseases to either enhance or diminish this function. Monoclonal antibodies and albumin-bound nanoparticles are examples of FcRn-dependent anti-cancer therapeutics. Despite its importance in drug delivery, little is known about FcRn expression in circulating immune cells. Through time-of-flight mass cytometry (CyTOF) we were able to characterize FcRn expression in peripheral blood mononuclear cell (PBMC) populations of pancreatic ductal adenocarcinoma (PDAC) patients and non-cancer donors. Furthermore, we were able to replicate these findings in an orthotopic murine model of PDAC. Altogether, we found that in both patients and mice with PDAC, FcRn was elevated in migratory and resident classical dendritic cell type 2 (cDC2) as well as monocytic and granulocytic myeloid-derived suppressor cell (MDSC) populations compared to tumor-free controls. Furthermore, PBMCs from PDAC patients had elevated monocyte, dendritic cells and MDSCs relative to non-cancer donor PBMCs. Future investigations into FcRn activity may further elucidate possible mechanisms of poor efficacy of antibody immunotherapies in patients with PDAC.

Abstract 2061: Deep immune profiling by mass cytometry revealed an association between the state of immune system before treatment and response to checkpoint inhibitor therapy in clear cell renal cell carcinoma

Abstract Checkpoint immunotherapy (CPI) and its combination with tyrosine kinase inhibitor (TKI) therapy are widely used in the treatment of many cancers, including clear cell renal cell carcinoma (ccRCC). Although recent advances in these therapies demonstrate effectiveness, a subset of patients are unresponsive. In this study, using deep immune profiling by mass cytometry (CyTOF), we analyzed peripheral blood mononuclear cell (PBMC) samples from 86 ccRCC patients pre- and post-treatment. The cohort included patients treated with first-line CPI (n = 43), TKI (n = 15) and combinatorial CPI+TKI (n = 28), with PBMC samples collected pre- (n = 41) and post- (n = 69) therapy (n = 24 patients total with paired samples). Samples were characterized by CyTOF analysis of 30 different cell surface markers. Using t-distributed stochastic neighbor embedding (tSNE) and Phenograph clustering, the presence of immune populations and subpopulations were characterized and associated with therapeutic response as well as longitudinally tracked. While most cell types did not differ between pre- and post-treatment states, a decrease in CD8 and CD4 PD1+ T cell subsets was observed in patients treated with CPI and CPI+TKI (p &amp;lt; 0.001). Also, increased CD8 effector memory T cells were observed in post-treatment samples in those treated with CPI or CPI+TKI (p &amp;lt; 0.001).Subsequent analysis of pre-treatment samples in the context of clinical outcome revealed specific features associated with favorable response to immunotherapy. Responders (R) in comparison with non-responders (NR) were characterized by an increased number of naive CD4 T cells (p = 0.03), CD8 T cells (p = 0.038), immature NK cells (p = 0.005) and lower percentage of granulocytes (p = 0.012). Using unsupervised hierarchical clustering, 8 patient groups were identified with a unique immune profile enriched by a certain PBMC population. Three groups (G1-G3), which included a high ratio of R, were enriched by naive T cells, immature NK cells and non-classical monocytes, respectively. The G4 group, enriched with B cells, consisted of R and NR in equal numbers. The G5-G8 groups, enriched with granulocytes, classical monocytes, mature NK-cells and effector CD8 T cells, respectively, were primarily comprised of NR. Patients with favorable pre-treatment immune profiles also belonged to favorable groups post-treatment, with a similar trend observed with unfavorable profiles. In conclusion, this analysis revealed a remarkable association between the initial immune status or “portrait” of the patient before therapy and subsequent therapeutic outcome as well as the development of therapeutic resistance. Citation Format: Svetlana Shishkova, Dmitry Tabakov, Evgeny Egorov, Akshaya Ramachandran, Yang Lyu, Krystle Nomie, Jessica Brown, Vladimir Zyrin, Alexander Zaytcev, Natalia Miheecheva, Ekaterina Postovalova, Alexander Bagaev, Ravshan Ataullakhanov, James Hsieh. Deep immune profiling by mass cytometry revealed an association between the state of immune system before treatment and response to checkpoint inhibitor therapy in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2061.