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ObjectiveAs the development and progression of colorectal cancer (CRC) are known to be affected by the immune system, cell subsets such as T cells, natural killer (NK) cells, and natural killer T (NKT) cells are considered interesting targets for immunotherapy and clinical biomarker research. Until now, the role of systemic immune profiles in tumor progression remains unclear. In this study, we aimed to characterize the immunophenotype of circulating T cells, NK cells, and NKT-like cells in patients with CRC, and to subsequently correlate these immunophenotypes to clinical follow-up data.MethodsUsing multiparameter flow cytometry, the subset distribution and immunophenotype of T cells (CD3+CD56−), CD56dim NK cells (CD3−CD56dim), CD56bright NK cells (CD3−CD56bright), and NKT-like (CD3+CD56+) cells were investigated in peripheral blood mononuclear cell (PBMC) samples from 71 CRC patients and 19 healthy donors.ResultsCRC patients showed profound differences in immune cell subset distribution and their immunophenotype compared to healthy donors, as characterized by increased percentage of regulatory T cells, and reduced expression level of the natural cytotoxicity receptors NKp44 and NKp46 on both CD56dim NK cells and NKT-like cells. Finally, we showed in a multivariate analysis that above-median percentage of CD16+ NKT-like cells was independently associated with shorter disease-free survival in CRC patients.ConclusionThe altered phenotype of circulating immune cell subsets in CRC and its association with clinical outcome highlight the potential use of PBMC subsets as prognostic biomarkers in CRC, thereby contributing to better insight into the role of systemic immune profiles in tumor progression.

In patients with nasopharyngeal carcinoma (NPC), the alteration of immune responses in peripheral blood remains unclear. In this study, we established an immune cell profile for patients with NPC and used flow cytometry and machine learning (ML) to identify the characteristics of this profile. After isolation of circulating leukocytes, the proportions of 104 immune cell subsets were compared between NPC group and the healthy control group (HC). Data obtained from the immune cell profile were subjected to ML training to differentiate between the immune cell profiles of the NPC and HC groups. We observed that subjects in the NPC group presented higher proportions of T cells, memory B cells, short‐lived plasma cells, IgG‐positive B cells, regulatory T cells, MHC II+ T cells, CTLA4+ T cells and PD‐1+ T cells than subjects in the HC group, indicating weaker and compromised cellular and humoral immune responses. ML revealed that monocytes, PD‐1+ CD4 T cells, memory B cells, CTLA4+ CD4 Treg cells and PD‐1+ CD8 T cells were strongly contributed to the difference in immune cell profiles between the NPC and HC groups. This alteration can be fundamental in developing novel immunotherapies for NPC.

Previous research has investigated highly inbred chicken genetic lines from a metabolic, immune response, genetic profile, and immune trait standpoint, including response to lipopolysaccharide (LPS). Fayoumi lines (M5.1, M15.2) are known for their resistance to bacterial and viral infections, while Leghorn lines (Ghs6, Ghs13) display lower disease resistance. Results highlighted a need to increase LPS dose above initial work using 1mg/kg bodyweight (BW). Therefore, this study investigated the immune profiles and metabolic phenotypes of peripheral blood mononuclear cells (PBMC) from highly inbred genetic lines under resting and stressed metabolic states. Fifty-four adult birds from 5 highly inbred genetic lines (M5.1, M15.2, Ghs13, Line-8, and Sp-21.1) were randomly assigned to 0.9% sterile saline control or 2.4 mg/kg BW intramuscular LPS ( Escherichia coli O55:B5). BW was recorded at baseline before injection and 24 h post-injection (hpi). Cloacal temperature was recorded at baseline, 2 hpi, and 24 hpi, while blood was collected for flow cytometry and metabolic analysis. Data were analyzed using the SAS 9.4 MIXED procedure with genetic line, injection status, and interaction as fixed effects, with significance at p ≤ 0.05. Baseline immune cell profiles varied by line ( p ≤ 0.001). At 2 hpi, LPS did not impact BW or temperature, but influenced all queried immune cell populations while decreasing ATP production and glycolytic rates ( p ≤ 0.02). At 2 hpi, M5.1, Line-8, and Sp-21.1 LPS-inoculated birds had increased circulating CD3 + cells (51.8-62.3%, p ≤ 0.0001). LPS decreased CD3 + CD1.1 + cell levels by 34.1% at 2 hpi ( p ≤ 0.0001). M5.1, M15.2, and Line-8 controls had 14.9-66.5% higher CD3 + CD4 + levels than LPS-inoculated birds, while CD3 + CD4 + cells were 12.2% lower in Ghs13 post-LPS ( p ≤ 0.0001). CD3 + CD8α + populations increased 41.1-63.2% in all LPS-injected birds at 2 hpi, except Ghs13 ( p ≤ 0.0001). These results highlight genetic line-specific immune responses to LPS. By 24 hpi, immune profiles and glycolytic rates were largely recovered from LPS, while genetic line effects persisted, indicating line-specific immune responses ( p ≤ 0.04). Further understanding cellular preference and metabolic switching during inflammatory challenges could provide insight into how to best support and optimize bird performance during the production cycles.

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 and translational models.

Background Combination immunotherapies, such as pembrolizumab plus lenvatinib (PL), are commonly used in treatment for unresectable hepatocellular carcinoma (uHCC). However, it remains challenging to predict which patients will benefit from this therapy. This study aimed to address this issue by comparing immune cell profiles (ICPs) between uHCC patients with objective response (responders, R) and those with tumor progression (non-responders, NR) following PL therapy, and to identify the key contributors to ICPs. Methods We prospectively enrolled 51 uHCC patients between July 2019 and July 2023. Peripheral blood samples were collected prior to initiating PL therapy, and ICPs were analyzed according to tumor response according to RECIST 1.1 criteria. A machine learning (ML) model was developed to differentiate R from NR using baseline ICP data. Results 16 patients achieved objective tumor responses, while 11 experienced disease progression following PL therapy. Responders exhibited higher levels of total T cells, CD8 T cells, and PD-1 + subpopulations of CD4 T cells, CD8 T cells, and NK cells. In contrast, NR had higher proportions of PD-L1 + monocytes. The trained ICP-based ML model accurately discriminated between the two groups, achieving 100% sensitivity and 66.7% specificity, with CD8 T cells, PD-1 + CD8 NK cells, and PD-L1 + monocytes contributing significantly to the classification. Conclusion This study recognized distinct ICPs between uHCC patients with and without tumor response to PL therapy and identified key contributing immune subpopulations. These findings provide a foundation for developing predictive tools for clinical outcomes before initiating combination immunotherapy.

There is increasing evidence for heterogeneity in type 1 diabetes mellitus (T1D): not only the age of onset and disease progression rate differ, but also the risk of complications varies markedly. Consequently, the presence of different disease endotypes has been suggested. Impaired T and B cell responses have been established in newly diagnosed diabetes patients. We hypothesized that deciphering the immune cell profile in peripheral blood of adults with longstanding T1D may help to understand disease heterogeneity. Adult patients with longstanding T1D and healthy controls (HC) were recruited, and their blood immune cell profile was determined using multicolour flow cytometry followed by a machine-learning based elastic-net (EN) classification model. Hierarchical clustering was performed to identify patient-specific immune cell profiles. Results were compared to those obtained in matched healthy control subjects. Hierarchical clustering analysis of flow cytometry data revealed three immune cell composition-based distinct subgroups of individuals: HCs, T1D-group-A and T1D-group-B. In general, T1D patients, as compared to healthy controls, showed a more active immune profile as demonstrated by a higher percentage and absolute number of neutrophils, monocytes, total B cells and activated CD4+CD25+ T cells, while the abundance of regulatory T cells (Treg) was reduced. Patients belonging to T1D-group-A, as compared to T1D-group-B, revealed a more proinflammatory phenotype characterized by a lower percentage of FOXP3+ Treg, higher proportions of CCR4 expressing CD4 and CD8 T cell subsets, monocyte subsets, a lower Treg/conventional Tcell (Tconv) ratio, an increased proinflammatory cytokine (TNFα, IFNγ) and a decreased anti-inflammatory (IL-10) producing potential. Clinically, patients in T1D-group-A had more frequent diabetes-related macrovascular complications. Machine-learning based classification of multiparameter flow cytometry data revealed two distinct immunological profiles in adults with longstanding type 1 diabetes; T1D-group-A and T1D-group-B. T1D-group-A is characterized by a stronger pro-inflammatory profile and is associated with a higher rate of diabetes-related (macro)vascular complications.

The interactions between T-cell chronic lymphoproliferative disorder (T-CLPD) tumor cells and the bystander immune cells may play a critical role in the failure of immune surveillance and disease progression, but the altered blood immune profiles of T-CLPD remain unknown. Here we analyzed the distribution of residual non-tumoral immune cells in blood of 47 T-CLPD patients -14 T-prolymphocytic leukemia (T-PLL), 7 Sézary syndrome/mycosis fungoides (SS/MF) and 26 T-large granular lymphocytic leukemia (T-LGLL)-, as tumor models of neoplastic T-cells that resemble naive/central memory (N/CM), memory and terminal effector T-cells, respectively, compared to 110 age- and sex-matched healthy donors, using spectral flow cytometry. Overall, our results showed deeply altered immune cell profiles in T-PLL, characterized by significantly increased counts of monocytes, dendritic cells, B-cells, NK-cells and innate lymphoid cells (ILC) -particularly ILC3-, together with reduced normal T-cells. In contrast, SS/MF showed neutrophilia, associated with decreased numbers of dendritic cells and NK-cells, potentially reflecting their increased migration from blood to the skin. In turn, T-LGLL displayed the mildest immune impairment, dependent on the TCD4+ . TCD8+ nature of the clonal T-cells and presence of mutations among TαβCD8+ T-LGLL cases. Further dissection of the normal T-cell compartment showed a significant reduction of the earliest T-cell maturation compartments (N/CM) in T-PLL and SS/MF, whereas T-cells remained within normal ranges in T-LGLL, with only a minor reduction of N/CM T-cells. These findings point out the existence of differentially altered innate and adaptive immune cell profiles in the distinct diagnostic subtypes of T-CLPD, with progressively less pronounced alterations from T-PLL and SS/MF to T-LGLL.

Utilize immune cell profiles in the cerebrospinal fluid (CSF) to advance the understanding and potentially support the diagnosis of inflammatory neuropathies. We analyzed CSF cell flow cytometry data of patients with definite Guillain-Barré syndrome (GBS, = 26) and chronic inflammatory demyelinating polyneuropathy (CIDP, = 32) based on established diagnostic criteria in comparison to controls with relapsing-remitting multiple sclerosis (RRMS, = 49) and idiopathic intracranial hypertension (IIH, = 63). Flow cytometry revealed disease-specific changes of CSF cell composition with a significant increase of NKT cells and CD8+ T cells in CIDP, NK cells in GBS, and B cells and plasma cells in MS in comparison to IIH controls. Principal component analysis demonstrated distinct CSF immune cells pattern in inflammatory neuropathies vs. RRMS. Systematic receiver operator curve (ROC) analysis identified NKT cells as the best parameter to distinguish GBS from CIDP. Composite scores combing several of the CSF parameters differentiated inflammatory neuropathies from IIH and GBS from CIDP with high confidence. Applying a novel dimension reduction technique, we observed an intra-disease heterogeneity of inflammatory neuropathies. Inflammatory neuropathies display disease- and subtype-specific alterations of CSF cell composition. The increase of NKT cells and CD8+ T cells in CIDP and NK cells in GBS, suggests a central role of cytotoxic cell types in inflammatory neuropathies varying between acute and chronic subtypes. Composite scores constructed from multi-dimensional CSF parameters establish potential novel diagnostic tools. Intra-disease heterogeneity suggests distinct disease mechanisms in subgroups of inflammatory neuropathies.

Aerosolized endotoxins such as Lipopolysaccharide (LPS), found in livestock environments, induce an inflammatory mediator cascade. Poultry are commonly exposed to LPS over the growth cycle; however, little is known regarding the cumulative impact of intramuscular LPS injection and its effects on immune cellular metabolism, pathway preferences, and clearance response. Utilizing a LPS model in chickens can offer insight into host immune responses and provide a better understanding of immune tolerance to this endotoxin and major component of Gram-negative bacteria. Therefore, the study objectives were to compare metabolic phenotypes and immune profiles of isolated peripheral blood mononuclear cells (PBMC) from two ages of adult White Leghorn roosters before and post-LPS injection. A total of 20 adult White Leghorn roosters aged 1 yr. or 2.5+ yrs. were randomly assigned to sterile saline or 1 mg/kg body weight LPS ( Escherichia coli O55:B5, LPS) injected intramuscularly across 4 sites in breast and thigh muscles. Body weight was recorded before injections at baseline and 24 h post-injection (hpi). Cloacal temperature and blood collections were performed at baseline, 6 hpi, and 24 hpi. PBMC were isolated for Agilent Seahorse XF metabolic analysis and multicolor flow cytometry. Plasma was collected for a C-reactive protein (CRP) enzyme-linked immunosorbent assay. Statistical analysis was performed using the MIXED procedure with fixed effects of age, injection status, and age X injection interaction followed by Tukey–Kramer adjustment (SAS 9.4), with significance denoted at p ≤ 0.05. Aged roosters were found to have fewer CD3 + CD8α + T cells at baseline compared to younger roosters ( p  < 0.05) while generally displaying delayed immunometabolic changes post-LPS injection compared to younger roosters. Young roosters administered LPS had significantly reduced CRP at 6 hpi compared to control, while aged roosters significantly increased CRP production by 24 hpi ( p  < 0.05). Both ages responded similarly to inhibitory assays, suggesting that the ability to respond was not different based on age. Overall, results suggest adult roosters may respond differently to LPS injection based on age and immune cell presence, likely due to accumulated exposure to LPS in poultry environments.

Lipopolysaccharide (LPS), a gram-negative bacterial cell wall component commonly used in animal models of inflammation, is also universally found in poultry environments. Documented LPS effects in production animals include reduced feed intake and weight loss; however, research into LPS’s impact on cellular metabolism and immune recovery is limited. This study compared baseline and stressed metabolic phenotypes of peripheral blood mononuclear cells (PBMC) from highly inbred genetic lines and examined fuel preference, cell profiles, and C-reactive protein (CRP) expression at baseline and post-LPS injection. Forty birds from 4 genetic lines (Ghs, Line-8, Sp-21.1, and AIL-F) were randomly assigned to 1 of 2 treatments, receiving intramuscular injections of saline or 1 mg/kg BW LPS ( Escherichia coli O55: B5). Body weight was recorded before injection (baseline) and 24 h post-injection (hpi), with cloacal temperature recorded at baseline, 6 hpi, and 24 hpi. Blood was collected at all timepoints for PBMC isolation, metabolic analysis, flow cytometry, and plasma CRP ELISA. Statistical analysis used the SAS 9.4 MIXED procedure with fixed effects of genetic line, injection status, and their interaction followed by Tukey–Kramer adjustment, with significance denoted at p  ≤ 0.05. Baseline immune profiles and ATP production varied by line ( p ≤ 0.02). LPS did not significantly impact body weight or temperature but influenced all immune cell populations and CRP concentration at 6 hpi ( p ≤ 0.02). Sp-21.1 exhibited a glycolytic metabolic profile and higher baseline CD3 + CD1.1 + and CD3 + CD4 + populations, suggesting enhanced antigen presentation and cytokine signaling. AIL-F displayed sustained monocyte/macrophage activation post-LPS and the highest baseline CD3 + CD8α + populations, indicating a distinct cytotoxic immune response. Line-8 maintained the highest CD3 + populations post-LPS and increased ATP production at 6 hpi, suggesting a balance between immune activation and metabolic compensation. Ghs exhibited a depletion of monocyte/macrophage + cells post-LPS but later recovered, highlighting a delayed immune response that may impact pathogen resistance. Results suggest genetic line may have a greater influence on metabolic pathway preferences than LPS injection in this experiment. Characterizing metabolic changes during immune activation and recovery may offer insights into breed-specific production traits and inform future breeding and management strategies to enhance health and production efficiency.