Causal role of immune cells in thyroid cancer: a bidirectional Mendelian randomization study.

Background: The immune system plays an important role in the development and treatment of thyroid cancer(THCA).However, the correlation between immune cells and THCA has not been systematically studied. Methods: This study used a two-sample Mendelian randomization (MR) study to determine the causal relationship between immune cell characteristics and THCA. Based on a large sample of publicly available genetic data, we explored the causal relationship between 731 immune cell characteristics and THCA risk. The 731 immunophenotypes were divided into 7 groups, including B cell panel(n=190),cDC panel(n=64),Maturation stages of T cell panel(n=79),Monocyte panel(n=43),Myeloid cell panel(n=64),TBNK panel(n=124),and Treg panel(n=167). The sensitivity of the results was analyzed, and heterogeneity and horizontal pleiotropy were excluded. Results: After FDR correction, the effect of immunophenotype on THCA was not statistically significant. It is worth mentioning, however, that there are some unadjusted low P-values phenotypes. The odds ratio (OR) of CD62L on monocyte on THCA risk was estimated to be 0.953 (95% CI=0.930~0.976, P=1.005×10-4),and which was estimated to be 0.975(95% CI=0.961-0.989, P=7.984×10-4) for Resting Treg%CD4 on THCA risk. Furthermore, THCA was associated with a reduced risk of 5 immunophenotype:CD25 on CD39+ CD4 on Treg (OR=0.871, 95% CI=0.812~0.935, P=1.274×10-4), activated Treg AC (OR=0.884, 95% CI=0.820~0.953, P=0.001), activated & resting Treg % CD4 Treg (OR=0.872, 95%CI=0.811~0.937,P=2.109×10-4),CD28- CD25++ CD8br AC(OR=0.867,95% CI=0.809~0.930,P=6.09×10-5),CD28-CD127-CD25++CD8brAC(OR=0.875,95%CI=0.814~0.942,P=3.619×10-4).THCA was associated with an increased risk of Secreting Treg % CD4 Treg (OR=1.143, 95% CI=1.064~1.229, P=2.779×10-4) and CD19 on IgD+ CD24+ (OR=1.118, 95% CI=1.041~1.120, P=0.002). Conclusions: These findings suggest the causal associations between immune cells and THCA by genetic means. Our results may have the potential to provide guidance for future clinical research.

Peripheral PD-1+NK cells could predict the 28-day mortality in sepsis patients.

Background: Unbalanced inflammatory response is a critical feature of sepsis, a life-threatening condition with significant global health burdens. Immune dysfunction, particularly that involving different immune cells in peripheral blood, plays a crucial pathophysiological role and shows early warning signs in sepsis. The objective is to explore the relationship between sepsis and immune subpopulations in peripheral blood, and to identify patients with a higher risk of 28-day mortality based on immunological subtypes with machine-learning (ML) model. Methods: Patients were enrolled according to the sepsis-3 criteria in this retrospective observational study, along with age- and sex-matched healthy controls (HCs). Data on clinical characteristics, laboratory tests, and lymphocyte immunophenotyping were collected. XGBoost and k-means clustering as ML approaches, were employed to analyze the immune profiles and stratify septic patients based on their immunological subtypes. Cox regression survival analysis was used to identify potential biomarkers and to assess their association with 28-day mortality. The accuracy of biomarkers for mortality was determined by the area under the receiver operating characteristic (ROC) curve (AUC) analysis. Results: The study enrolled 100 septic patients and 89 HCs, revealing distinct lymphocyte profiles between the two groups. The XGBoost model discriminated sepsis from HCs with an area under the receiver operating characteristic curve of 1.0 and 0.99 in the training and testing set, respectively. Within the model, the top three highest important contributions were the percentage of CD38+CD8+T cells, PD-1+NK cells, HLA-DR+CD8+T cells. Two clusters of peripheral immunophenotyping of septic patients by k-means clustering were conducted. Cluster 1 featured higher proportions of PD1+ NK cells, while cluster 2 featured higher proportions of naïve CD4+T cells. Furthermore, the level of PD-1+NK cells was significantly higher in the non-survivors than the survivors (15.1% vs 8.6%, P<0.01). Moreover, the levels of PD1+ NK cells combined with SOFA score showed good performance in predicting the 28-day mortality in sepsis (AUC=0.91,95%CI 0.82-0.99), which is superior to PD1+ NK cells only(AUC=0.69, sensitivity 0.74, specificity 0.64, cut-off value of 11.25%). In the multivariate Cox regression, high expression of PD1+ NK cells proportion was related to 28-day mortality (aHR=1.34, 95%CI 1.19 to 1.50; P<0.001). Conclusion: The study provides novel insights into the association between PD1+NK cell profiles and prognosis of sepsis. Peripheral immunophenotyping could potentially stratify the septic patients and identify those with a high risk of 28-day mortality.

Detailed phenotyping reveals diverse and highly skewed neutrophil subsets in both the blood and airways during active tuberculosis infection.

Introduction: Neutrophils play a complex and important role in the immunopathology of TB. Data suggest they are protective during early infection but become a main driver of immunopathology if infection progresses to active disease. Neutrophils are now recognized to exist in functionally diverse states, but little work has been done on how neutrophil states or subsets are skewed in TB disease. Methods: To address this, we carried out comprehensive phenotyping by flow cytometry of neutrophils in the blood and airways of individuals with active pulmonary TB with and without HIV co-infection recruited in Durban, South Africa. Results: Active TB was associated with a profound skewing of neutrophils in the blood toward phenotypes associated with activation and apoptosis, reduced phagocytosis, reverse transmigration, and immune regulation. This skewing was also apparently in airway neutrophils, particularly the regulatory subsets expressing PDL-1 and LOX-1. HIV co-infection did not impact neutrophil subsets in the blood but was associated with a phenotypic change in the airways and a reduction in key neutrophil functional proteins cathelicidin and arginase 1. Discussion: Active TB is associated with profound skewing of blood and airway neutrophils and suggests multiple mechanisms by which neutrophils may exacerbate the immunopathology of TB. These data indicate potential avenues for reducing neutrophil-mediated lung pathology at the point of diagnosis.

Causal association between immune cells and lung cancer risk: a two-sample bidirectional Mendelian randomization analysis.

Background: Previous studies have highlighted the crucial role of immune cells in lung cancer development; however, the direct link between immunophenotypes and lung cancer remains underexplored. Methods: We applied two-sample Mendelian randomization (MR) analysis, using genetic variants as instruments to determine the causal influence of exposures on outcomes. This method, unlike traditional randomized controlled trials (RCTs), leverages genetic variants inherited randomly at conception, thus reducing confounding and preventing reverse causation. Our analysis involved three genome-wide association studies to assess the causal impact of 731 immune cell signatures on lung cancer using genetic instrumental variables (IVs). We initially used the standard inverse variance weighted (IVW) method and further validated our findings with three supplementary MR techniques (MR-Egger, weighted median, and MR-PRESSO) to ensure robustness. We also conducted MR-Egger intercept and Cochran's Q tests to assess heterogeneity and pleiotropy. Additionally, reverse MR analysis was performed to explore potential causality between lung cancer subtypes and identified immunophenotypes, using R software for all statistical calculations. Results: Our MR analysis identified 106 immune signatures significantly associated with lung cancer. Notably, we found five suggestive associations across all sensitivity tests (P<0.05): CD25 on IgD- CD24- cells in small cell lung carcinoma (ORIVW =0.885; 95% CI: 0.798-0.983; P IVW =0.022); CD27 on IgD+ CD24+ cells in lung squamous cell carcinoma (ORIVW =1.054; 95% CI: 1.010-1.100; P IVW =0.015); CCR2 on monocyte cells in lung squamous cell carcinoma (ORIVW =0.941; 95% CI: 0.898-0.987; P IVW =0.012); CD123 on CD62L+ plasmacytoid dendritic cells (ORIVW =0.958; 95% CI: 0.924-0.992; P IVW =0.017) as well as on plasmacytoid dendritic cells (ORIVW =0.958; 95% CI: 0.924-0.992; P IVW =0.017) in lung squamous cell carcinoma. Conclusion: This study establishes a significant genomic link between immune cells and lung cancer, providing a robust basis for future clinical research aimed at lung cancer management.

Effects of Injectable Solutions on the Quality of Monocyte-Derived Dendritic Cells for Immunotherapy.

Therapeutic vaccines based on monocyte-derived dendritic cells have been shown to be promising strategies and may act as complementary treatments for viral infections, cancers, and, more recently, autoimmune diseases. Alpha-type-1-polarized dendritic cells (aDC1s) have been shown to induce type-1 immunity with a high capacity to produce interleukin-12p70 (IL-12p70). In the clinical use of cell-based therapeutics, injectable solutions can affect the morphology, immunophenotypic profile, and viability of cells before delivery and their survival after injection. In this sense, preparing a cell suspension that maintains the quality of aDC1s is essential to ensure effective immunotherapy. In the present study, monocytes were differentiated into aDC1s in the presence of IL-4 and GM-CSF. On day 5, the cells were matured by the addition of a cytokine cocktail consisting of IFN-α, IFN-γ, IL-1ß, TNF-α, and Poly I:C. After 48 hr, mature aDC1s were harvested and suspended in two different solutions: normal saline and Ringer's lactate. The maintenance of cells in suspension was evaluated after 4, 6, and 8 hr of storage. Cell viability, immunophenotyping, and apoptosis analyses were performed by flow cytometry. Cellular morphology was observed by electron microscopy, and the production of IL-12p70 by aDC1s was evaluated by ELISA. Compared with normal saline, Ringer's lactate solution was more effective at maintaining DC viability for up to 8 hr of incubation at 4 or 22°C.

Comparative immune profiling of pancreatic ductal adenocarcinoma progression among South African patients.

BACKGROUND: Pancreatic Ductal Adenocarcinoma (PDAC) is an aggressive cancer characterized by an immunosuppressive microenvironment. Patients from specific ethnicities and population groups have poorer prognoses than others. Therefore, a better understanding of the immune landscape in such groups is necessary for disease elucidation, predicting patient outcomes and therapeutic targeting. This study investigated the expression of circulating key immune cell markers in South African PDAC patients of African ancestry. METHODS: Blood samples were obtained from a total of 6 healthy volunteers (HC), 6 Chronic Pancreatitis (CP) and 34 PDAC patients consisting of 22 resectable (RPC), 8 locally advanced (LAPC) and 4 metastatic (MPC). Real-time Quantitative Polymerase Chain reactions (RT-qPCR), Metabolomics, Enzyme-Linked Immunosorbent Assay (ELISA), Reactive Oxygen Species (ROS), and Immunophenotyping assays were conducted. Statistical analysis was conducted in R (v 4.3.2). Additional analysis of single-cell RNA data from 20 patients (16 PDAC and 4 controls) was conducted to interrogate the distribution of T-cell and Natural Killer cell populations. RESULTS: Granulocyte and neutrophil levels were significantly elevated while lymphocytes decreased with PDAC severity. The total percentages of CD3 T-cell subpopulations (helper and double negative T-cells) decreased when compared to HC. Although both NK (p = 0.014) and NKT (p < 0.001) cell levels increased as the disease progressed, their subsets: NK CD56dimCD16- (p = 0.024) and NKTs CD56+ (p = 0.008) cell levels reduced significantly. Of note is the negative association of NK CD56dimCD16- (p < 0.001) cell levels with survival time. The gene expression analyses showed no statistically significant correlation when comparing the PDAC groups with the controls. The inflammatory status of PDAC was assessed by ROS levels of serum which were elevated in CP (p = 0.025), (RPC (p = 0.003) and LAPC (p = 0.008)) while no significant change was observed in MPC, compared to the HC group. ROS was shown to be positively correlated with GlycA (R = 0.45, p = 0.0096). Single-cell analyses showed a significant difference in the ratio of NKT cells per total cell counts in LAPC (p < 0.001) and MPC (p < 0.001) groups compared with HC, confirming observations in our sample group. CONCLUSION: The expression of these immune cell markers observed in this pilot study provides insight into their potential roles in tumour progression in the patient group and suggests their potential utility in the development of immunotherapeutic strategies.

Controlled human hookworm infection remodels plasmacytoid dendritic cells and regulatory T cells towards profiles seen in natural infections in endemic areas.

Hookworm infection remains a significant public health concern, particularly in low- and middle-income countries, where mass drug administration has not stopped reinfection. Developing a vaccine is crucial to complement current control measures, which necessitates a thorough understanding of host immune responses. By leveraging controlled human infection models and high-dimensional immunophenotyping, here we investigated the immune remodeling following infection with 50 Necator americanus L3 hookworm larvae in four naïve volunteers over two years of follow-up and compared the profiles with naturally infected populations in endemic areas. Increased plasmacytoid dendritic cell frequency and diminished responsiveness to Toll-like receptor 7/8 ligand were observed in both controlled and natural infection settings. Despite the increased CD45RA+ regulatory T cell (Tregs) frequencies in both settings, markers of Tregs function, including inducible T-cell costimulatory (ICOS), tumor necrosis factor receptor 2 (TNFR2), and latency-associated peptide (LAP), as well as in vitro Tregs suppressive capacity were higher in natural infections. Taken together, this study provides unique insights into the immunological trajectories following a first-in-life hookworm infection compared to natural infections.

Causal role of immune cells in uveitis: Mendelian randomization study.

Background: Uveitis, characterized by inflammation of the iris, ciliary body, and choroid, presents a significant global clinical challenge, contributing substantially to visual impairment. Risk factors include autoimmune diseases and immune cell dysfunctions, yet many remain unidentified. Immune cells, notably T cells, B cells, and monocytes, play pivotal roles in uveitis pathogenesis. While biologic agents show promise, comprehensive studies on immune cell types in ocular diseases are lacking. Genome-wide association studies (GWAS) and Mendelian randomization (MR) present promising avenues to elucidate genetic susceptibilities and causal relationships between immune cell traits and uveitis risk. Methods: Two-sample MR analysis was used to evaluate the causal relationship between 731 immune cells and uveitis, and genome-wide significance analysis was performed for genetic variation in 731 immune cells traits (P < 5 × 10-8). Immune characteristics include median fluorescence intensity (MFI), relative cell counts (RC), absolute cell counts (AC), and morphological parameters (MP), which were determined by published GWAS, and public data from the IEU Open GWAS database. The main analysis method of MR is inverse variance weighting (IVW). Heterogeneity and horizontal pleiotropy were also assessed. Results: 5 immunophenotypes, including CD62L-DC %DC, IgD+ CD38dim %B cell, CD3 on CM CD4+T cell, CD3 on CD45RA-CD4 +T cell, and CD3 on CD39+ CD4+ Treg may increase the risk of uveitis. 5 immunophenotypes, including CD11b on CD33dim HLA DR-Myeloid cell, HLA DR on CD33dim HLA DR+ CD11b-myeloid cell, CD14-CD16 + %monocyte, HLA DR on CD14-CD16 + monocyte and PDL-1 on CD14-CD16 + monocyte was negatively associated with the risk of uveitis. Among them, HLA DR on CD14-CD16 + monocyte (OR=0.921, 95%CI =0.875-0.970, P=0.001) and HLA DR on CD33dim HLA DR+ CD11b- (OR=0.879, 95%CI = 0.833-0.927, P=0.00) were negatively associated with the risk of uveitis in bi-direction. Conclusion: These results indicate that 10 immune cells traits are significantly associated with the risk of developing uveitis and 2 of them were strongly associated with uveitis bi-directionally, after excluding the effects of confounding factors such as some immune diseases, which provided new ideas and therapeutic targets for the study of immune mechanism of uveitis.

A novel NKp80-based strategy for universal identification of normal, reactive and tumor/clonal natural killer-cells in blood.

Purpose: Natural killer (NK) cells are traditionally identified by flow cytometry using a combination of markers (CD16/CD56/CD3), because a specific NK-cell marker is still missing. Here we investigated the utility of CD314, CD335 and NKp80, compared to CD16/CD56/CD3, for more robust identification of NK-cells in human blood, for diagnostic purposes. Methods: A total of 156 peripheral blood (PB) samples collected from healthy donors (HD) and patients with diseases frequently associated with loss/downregulation of classical NK-cell markers were immunophenotyped following EuroFlow protocols, aimed at comparing the staining profile of total blood NK-cells for CD314, CD335 and NKp80, and the performance of distinct marker combinations for their accurate identification. Results: NKp80 showed a superior performance (vs. CD314 and CD335) for the identification of NK-cells in HD blood. Besides, NKp80 improved the conventional CD16/CD56/CD3-based strategy to identify PB NK-cells in HD and reactive processes, particularly when combined with CD16 for further accurate NK-cell-subsetting. Although NKp80+CD16 improved the identification of clonal/tumor NK-cells, particularly among CD56- cases (53%), aberrant downregulation of NKp80 was observed in 25% of patients, in whom CD56 was useful as a complementary NK-cell marker. As NKp80 is also expressed on T-cells, we noted increased numbers of NKp80+ cytotoxic T-cells at the more advanced maturation stages, mostly in adults. Conclusion: Here we propose a new robust approach for the identification of PB NK-cells, based on the combination of NKp80 plus CD16. However, in chronic lymphoproliferative disorders of NK-cells, addition of CD56 is recommended to identify clonal NK-cells, due to their frequent aberrant NKp80- phenotype.

Immune cells phenotype and bioenergetic measures in CD4+ T cells differ between high and low feed efficient dairy cows.

Identifying sources of variance that contribute to residual feed intake (RFI) can aid in improving feed efficiency. The objectives of this study were to investigate immune cells phenotype and bioenergetic measures in CD4+ T cells in low feed efficient (LE) and high feed efficient (HE) dairy cows. Sixty-four Holstein cows were enrolled at 93 ± 22 days in milk (DIM) and monitored for 7 weeks to measure RFI. Cows with the highest RFI (LE; n = 14) or lowest RFI (HE; n = 14) were selected to determine immune cells phenotype using flow cytometry. Blood was sampled in the same LE and HE cows at 234 ± 22 DIM to isolate peripheral blood mononuclear cells, followed by magnetic separation of CD4+ T lymphocytes using bovine specific monoclonal antibodies. The metabolic function of isolated CD4+ T lymphocytes was evaluated under resting and activated states. An increased expression of CD62L+ cells within CD8+ T lymphocytes and CD21+ B lymphocytes was observed in HE cows compared to LE cows. CD4+ T lymphocytes of HE cows exhibited an increased mitochondrial and glycolytic activity in resting and activated states compared to LE cows. These data suggest that immune cells in HE cows exhibit an increased metabolic function, which might influence nutrient partitioning and utilization and serve as a source of variation in feed efficiency that warrants future investigation.

Evaluation of immunophenotypic alterations of peripheral blood lymphocytes and their sub-sets in uncomplicated P. Falciparum infection.

BACKGROUND: Malaria is a life-threatening parasitic disease typically transmitted through the bite of an infected Anopheles mosquito. There is ample evidence showing the potential of malaria infection to affect the counts of lymphocyte subpopulations in the peripheral blood, but the extent of alteration might not be consistent in all geographical locations, due to several local factors. Although Ghana is among the malaria-endemic countries, there is currently no available data on the level of alterations that occur in the counts of lymphocyte subpopulations during P. falciparum malaria infection among adults. AIM: The study was to determine the immunophenotypic alterations in the level of peripheral blood lymphocytes and their subsets in adults with uncomplicated P. falciparum malaria infection and apparently healthy participants. METHODS: The study was a cross-sectional comparative study conducted in two municipalities of the Volta region of Ghana. Blood samples were collected from study participants and taken through serology (P. falciparum/Pan Rapid Diagnostic Kits), microscopy (Thick and thin blood films) and Haematological (Flow cytometric and Full blood count) analysis. RESULTS: A total of 414 participants, comprising 214 patients with malaria and 200 apparently healthy individuals (controls) were recruited into this study. Parasite density of the malaria patients ranged from 75/µL to 84,364/µL, with a mean of 3,520/µL. It was also observed that the total lymphocytes slightly decreased in the P. falciparum-infected individuals (Mean ± SD: 2.08 ± 4.93 × 109/L) compared to the control group (Mean ± SD: 2.47 ± 0.80 × 109/L). Again, there was a significant moderate positive correlation between parasite density and haematocrit levels (r = 0.321, p < 0.001). Apart from CD45 + T-cells, more people in the control group had normal values for the lymphocyte subsets measured compared to the malaria patients. CONCLUSIONS: From the results obtained, there was high parasite density among the malaria patients suggestive of high intensity of infection in the case group. The malaria patients again showed considerable haematological alterations in lymphocyte sub-sets and the parasite density appeared to be strongly associated with CD4 + T-cell reduction. Also, the parasite density significantly associated with decreasing haematocrit levels. This indicates that lymphocyte subset enumeration can be used to effectively support malaria diagnosis.

[Clinicopathological features of primary mucosal CD30-positive T-cell lymphoproliferative disorders].

Objective: To investigate the clinicopathological features and differential diagnosis of primary mucosal CD30-positive T-cell lymphoproliferative disorders (pmCD30+TLPD). Methods: Eight cases of pmCD30+TLPD diagnosed from 2013 to 2023 at the Department of Pathology, Beijing Friendship Hospital Affiliated to Capital Medical University and Beijing Ludaopei Hospital were retrospectively collected. The immunophenotype, EBV infection status and T-cell receptor (TCR) clonability of tumor cells were examined. The clinicopathological features were analyzed and related literatures were reviewed. Results: There were 5 females and 3 males, aged 28 to 73 years, without B symptoms, lack of trauma and autoimmune diseases. Seven cases occurred in oral mucosa and one in anal canal mucosa. Submucosal nodules with ulcerations were presented in all cases except one, which only submucosal nodule. Morphologically, there was different distribution of allotypic lymphocytes in inflammatory background. Four cases showed "kidney-shaped", "embryonic" and "horseshoe-shaped" cells, and one case resembled Hodgkin and Reed/Sternberg (HRS) cells. Allotypic lymphocytes expressed CD3 (7/8), CD4+/CD8-(7/8) and CD4-/CD8-(1/8). CD30 was uniformly strongly positive while ALK and CD56 were negative. In situ hybridization of EBER was negative in five cases (5/5). Clonal TCR gene rearrangement was positive in two cases. Four patients did not receive radiotherapy or chemotherapy. All the seven patients survived without disease except one died due to concurrent leukopenia. Conclusions: pmCD30+TLPD had a broad morphological spectrum and could be easily confused with primary cutaneous CD30+TLPD and systemic ALK-negative anaplastic large cell lymphoma involving mucosa, which may lead to misdiagnosis. Although the majority of the cases had a favorable prognosis, a few cases relapsed or progressed to lymphoma.

Phenotypic characterisation of bovine alveolar macrophages reveals two major subsets with differential expression of CD163.

Bovine alveolar macrophages (AMs) defend the lungs against pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis. However, little is known about the surface molecules expressed by bovine AMs and whether there is heterogeneity within the population. The purpose of this study was to characterise the bovine AM cell surface phenotype using flow cytometry. Bronchoalveolar lavage samples from four different calves were stained with a combination of antibodies against immune cell molecules prior to flow cytometric analysis. To assess the degree of expression, we considered the distribution and relative intensities of stained and unstained cells. We demonstrated that bovine AMs have high expression of CD172a, ADGRE1, CD206, and CD14, moderate expression of CD80, MHC II, CD1b, and CD40, low expression of CX3CR1 and CD86, and little or no expression of CD16 and CD26. Two distinct subsets of bovine AMs were identified based on CD163 expression. Subsequent analysis showed that the CD163+ subset had greater expression of other typical macrophage molecules compared to the CD163- subset, suggesting that these cells may perform different roles during infection. The characterisation of the uninfected bovine AM phenotype will provide a foundation for the examination of M. bovis-infected AMs.

Ultrasound-Guided Fine Needle Biopsy of Human Axillary Lymph Nodes to Assess B Cell Responses to Vaccination.

Ultrasound-guided fine needle biopsy, also known as fine needle aspiration, of human axillary lymph nodes is a safe and effective procedure to assess the immune response within the lymph nodes following vaccination. Once acquired, lymph node cells can be characterized via flow cytometric immunophenotyping and/or single-cell RNA sequencing for gene expression and T and B cell receptors. Analysis of the immune cells from the lymph nodes enables the investigation of T and B cells that may interact at this site. These interactions may lead to germinal center formation and expansion, critical for the generation of effective immunity to vaccination. Directly studying the dynamic processes and interaction of the key cells has been challenging in humans due to the anatomically protected location of these cells. Here, we describe the methods involved in ultrasound-guided fine needle biopsy of human axillary lymph nodes in response to vaccination and subsequent analyses of the B cell populations.

Comprehensive Flow Cytometric, Immunohistologic, and Molecular Assessment of Thymus Function in Rhesus Macaques.

The critical importance of the thymus for generating new naive T cells that protect against novel infections and are tolerant to self-antigens has led to a recent revival of interest in monitoring thymic function in species other than humans and mice. Nonhuman primates such as rhesus macaques (Macaca mulatta) provide particularly useful animal models for translational research in immunology. In this study, we tested the performance of a 15-marker multicolor Ab panel for flow cytometric phenotyping of lymphocyte subsets directly from rhesus whole blood, with validation by thymectomy and T cell depletion. Immunohistochemical and multiplex RNA expression analysis of thymus tissue biopsies and molecular assays on PBMCs were used to further validate thymus function. Results identify Ab panels that can accurately classify rhesus naive T cells (CD3+CD45RA+CD197+ or CD3+CD28+CD95-) and recent thymic emigrants (CD8+CD28+CD95-CD103+CD197+) using just 100 µl of whole blood and commercially available fluorescent Abs. An immunohistochemical panel reactive with pan-cytokeratin (CK), CK14, CD3, Ki-67, CCL21, and TdT provides histologic evidence of thymopoiesis from formalin-fixed, paraffin-embedded thymus tissues. Identification of mRNAs characteristic of both functioning thymic epithelial cells and developing thymocytes and/or molecular detection of products of TCR gene rearrangement provide additional complementary methods to evaluate thymopoiesis, without requiring specific Abs. Combinations of multiparameter flow cytometry, immunohistochemistry, multiplex gene expression, and TCR excision circle assays can comprehensively evaluate thymus function in rhesus macaques while requiring only minimal amounts of peripheral blood or biopsied thymus tissue.

Peripheral T Cell Development and Immunophenotyping of Twins with Heterozygous FOXN1 Mutations.

The transcription factor FOXN1 plays an established role in thymic epithelial development to mediate selection of maturing thymocytes. Patients with heterozygous loss-of-function FOXN1 variants are associated with T cell lymphopenia at birth and low TCR excision circles that can ultimately recover. Although CD4+ T cell reconstitution in these patients is not completely understood, a lower proportion of naive T cells in adults has suggested a role for homeostatic proliferation. In this study, we present an immunophenotyping study of fraternal twins with low TCR excision circles at birth. Targeted primary immunodeficiency testing revealed a heterozygous variant of uncertain significance in FOXN1 (c.1205del, p.Pro402Leufs*148). We present the immune phenotypes of these two patients, as well as their father who carries the same FOXN1 variant, to demonstrate an evolving immune environment over time. While FOXN1 haploinsufficiency may contribute to thymic defects and T cell lymphopenia, we characterized the transcriptional activity and DNA binding of the heterozygous FOXN1 variant in 293T cells and found the FOXN1 variant to have different effects across several target genes. These data suggest multiple mechanisms for similar FOXN1 variants pathogenicity that may be mutation specific. Increased understanding of how these variants drive transcriptional regulation to impact immune cell populations will guide the potential need for therapeutics, risk for infection or autoimmunity over time, and help inform clinical decisions for other variants that might arise.

Acute myeloid leukemia with ETV6::CHIC2 fusion gene: 'Pitfalls' in diagnosis.

OBJECTIVES: Acute myeloid leukemia (AML) with ETV6::CHIC2 and basophilia is rare in hematologic malignancies with poor prognosis. Due to the small number of clinical cases, it is misdiagnosed and missed frequently, and it is necessary to explore laboratory detection for accurate diagnosis. METHODS: We report a case of AML with ETV6::CHIC2 and basophilia by morphological screening, immunotyping with precise gating, interpretation of FISH results, and RNA transcriptome sequencing, thus laying the accurate diagnosis for clinical treatment. RESULTS: We confirmed a rare case of AML with ETV6::CHIC2 rather than FIP1L1::PDGFRA by morphological analysis, correct immunophenotyping via precise gating, rejecting one-sided view of FISH positive result and targeted RNA sequencing. Precise analysis and more advanced means avoid misdiagnosis and missed frequently. After accurate diagnosis, venetoclax and decitabine therapy were given instead of imatinib; eventually, the patient achieved a relatively good effect. DISCUSSION: Immunophenotype analysis is necessary to detect the expression of CD7 when encountering pseudo-lymphocytes with multilineage dysplasia and basophilia. FISH and RT-PCR are still indispensable means of diagnosis of fusion genes in hematologic malignancies but can only detect a limited number of known partner genes and fusion genes with known break points. NGS can achieve sequence analysis indiscriminately and detect all fusion transcripts theoretically, greatly improving the detection range. NGS sequencing is required for t(4;12)(q11;p13) in AML that are not accompanied by eosinophilia.

Training immunophenotyping deep learning models with the same-section ground truth cell label derivation method improves virtual staining accuracy.

Introduction: Deep learning (DL) models predicting biomarker expression in images of hematoxylin and eosin (H&E)-stained tissues can improve access to multi-marker immunophenotyping, crucial for therapeutic monitoring, biomarker discovery, and personalized treatment development. Conventionally, these models are trained on ground truth cell labels derived from IHC-stained tissue sections adjacent to H&E-stained ones, which might be less accurate than labels from the same section. Although many such DL models have been developed, the impact of ground truth cell label derivation methods on their performance has not been studied. Methodology: In this study, we assess the impact of cell label derivation on H&E model performance, with CD3+ T-cells in lung cancer tissues as a proof-of-concept. We compare two Pix2Pix generative adversarial network (P2P-GAN)-based virtual staining models: one trained with cell labels obtained from the same tissue section as the H&E-stained section (the 'same-section' model) and one trained on cell labels from an adjacent tissue section (the 'serial-section' model). Results: We show that the same-section model exhibited significantly improved prediction performance compared to the 'serial-section' model. Furthermore, the same-section model outperformed the serial-section model in stratifying lung cancer patients within a public lung cancer cohort based on survival outcomes, demonstrating its potential clinical utility. Discussion: Collectively, our findings suggest that employing ground truth cell labels obtained through the same-section approach boosts immunophenotyping DL solutions.

Pilot Study for Isolation of Stromal Vascular Fraction with Collagenase Using an Automated Processing System.

There are many potential therapeutic applications for autologous adipose-derived stromal cells. These cells are found in a heterogeneous population isolated from adipose tissue called the stromal vascular fraction (SVF). Closed automated systems are available to release cells from the adherent stroma. Here, we test one system to evaluate the heterogeneous output for yield, purity, cellular characterization, and stemness criteria. The SVF was isolated from three donors using the Automated Cell Station (ACS) from BSL Co., Ltd., Busan, Republic of Korea. The SVF cellular output was characterized for cell yield and viability, immunophenotyping analysis, pluripotent differentiation potential, adhesion to plastic, and colony-forming units. Additionally, the SVF was tested for endotoxin and collagenase residuals. The SVF yield from the ACS system was an average volume of 7.9 ± 0.5 mL containing an average of 19 × 106 nucleated cells with 85 ± 12% viability. Flow cytometry identified a variety of cells, including ASCs (23%), macrophages (24%), endothelial cells (5%), pericytes (4%), and transitional cells (0.5%). The final concentrated product contained cells capable of differentiating into adipogenic, chondrogenic, and osteogenic phenotypes. Furthermore, tests for SVF sterility and purity showed no evidence of endotoxin or collagenase residuals. The ACS system can efficiently process cells from adipose tissue within the timeframe of a single surgical procedure. The cellular characterization indicated that this system can yield a sterile and concentrated SVF output, providing a valuable source of ASCs within the heterogeneous cell population.

Blood immune cell profiling in adults with longstanding type 1 diabetes is associated with macrovascular complications.

Aims/hypothesis: 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. Methods: 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. Results: 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. Conclusions: 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.