Borrelia burgdorferi is strong inducer of IFN-γ production by human primary NK cells.

Natural Killer (NK) cells belong to the innate lymphoid lineage and are highly present in the human skin. NK cells can produce a range of pro-inflammatory mediators, including cytokines and chemokines. The role of NK(-T) cells in the immune response towards Borrelia burgdorferi infection was studied. The production of interleukin (IL)-6, IL-1ß and interferon-gamma (IFN-γ) by human primary peripheral blood mononuclear cells (PBMCs) exposed to B. burgdorferi was assessed. Interestingly, CD56+ (NK + NK-T) cells were the only cells within the PBMC-fraction that produced IFN-γ during the first 24 h of stimulation. Within the NK(-T) cell fraction, NK cells seemed to be responsible for the IFN-γ production. Since it was previously demonstrated that both TLR2 and NOD2 receptors are involved in the recognition of B. burgdorferi, the expression of both TLR2 and NOD2 mRNA on NK cells was determined. In contrast to TLR2, NOD2 mRNA was upregulated on CD56+ (NK + NK-T) cells after Borrelia exposure. Finally, to unravel the mechanisms underlying erythema migrans (EM) development, crosstalk between CD56+ (NK + NK-T) cells and keratinocytes was investigated. CD56+ (NK + NK-T) cells activated by B. burgdorferi produced soluble mediators strongly inducing the expression of antimicrobial peptides, such as ß-defensin-2 and psoriasin in human keratinocytes. In conclusion, CD56+ (NK + NK-T) cells produced IFN-γ shortly after exposure to B. burgdorferi and released soluble mediators that were able to activate keratinocytes. These observations underscore the important role of CD56+ (NK + NK-T) cells during early host defence when Borrelia burgdorferi enters the human skin during a tick bite.

Peripheral blood natural killer cells in sarcoidosis are associated with early cardiac involvement.

AIM: To evaluate the distribution of circulating immune cell subsets in peripheral blood of patients with sarcoidosis and investigate if there is an association with an underlying cardiac involvement. METHODS AND RESULTS: Eighty-five newly diagnosed treatment-naïve patients with sarcoidosis (50 women) were included in the study. All patients underwent a thorough cardiac investigation, including cardiac magnetic resonance imaging (CMR). Of all patients, 19 (23.53%) had myocardial involvement, and the NK subpopulation in these patients in peripheral blood was significantly decreased compared to patients without (n = 63, p = 0.001 and p = 0.003 respectively). The absolute number of NKT cells (CD3+CD16/56+ ) in patients with cardiac involvement was highly correlated with T2 map increased values in MRI (r = -686, p = 0.041) showing that low NKT cell count correlates with the inflammatory process of the heart. No difference in CD19, CD3, CD4, CD8 and CD3- NK cell counts was found between groups. Lung severity was not found to correlate with the number of NK cells. CONCLUSION: We found that low NK cell count in peripheral blood of patients with sarcoidosis is associated with cardiac involvement, and the number of NK-T cells correlates with CMR findings indicative of myocardial inflammation. This finding might have a potential clinical application in detecting clinically silent cardiac involvement in sarcoidosis and may also suggest potential targets for therapeutic interventions.

Increased Foxp3+Helios+ Regulatory T Cells and Decreased Acute Graft-versus-Host Disease after Allogeneic Bone Marrow Transplantation in Patients Receiving Sirolimus and RGI-2001, an Activator of Invariant Natural Killer T Cells.

Regulatory T (Treg) cells play a central role in immune tolerance and prevention of aberrant immune responses. Several studies have suggested that the risk of graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT) can be ameliorated by increasing Tregs. We have developed an approach of in vivo expansion of Tregs with RGI-2001, a novel liposomal formulation of a synthetic derivative of alpha-galactosylceramide, a naturally occurring ligand that binds to CD1 and activates and expands invariant natural killer cells. In preclinical studies, a single intravenous infusion of RGI-2001 expanded Treg and could ameliorate GVHD in a mouse model of allogeneic HCT. To explore the role of RGI-2001 in clinical HCT, we initiated a phase 2A clinical trial (n = 29), testing 2 different doses of RGI-2001 administered as a single infusion on day 0 of allogeneic HCT. RGI-2001 was well tolerated and without infusion reactions or cytokine release syndrome. A subset of patients (8 of 29, 28%) responded to RGI-2001 by inducing a markedly increased number of cells with a Treg phenotype. The Treg had a high Ki-67 index and were almost exclusively Helios+ and Foxp3+, indicating that their accumulation was due to expansion of natural Treg. Notably, the incidence of grade 2 to 4 GVHD in the 8 patients who responded to RGI-2001 was 12.5%, compared with 52.4% in the 21 patients who did not respond. No grade 3 or 4 GVHD was observed in the responder group, compared with a 9.5% incidence among nonresponders. Immunosuppression with sirolimus was also associated with a low incidence of GVHD, suggesting that RGI-2001 may have synergized with sirolimus to promote Treg expansion.

Potential non-T cells source of interleukin-4 in food allergy.

BACKGROUND: Recently, a study from the Consortium of Food Allergy Research (CoFAR) showed that allergen-induced IL-4 expression in CD25(+) mononuclear cells was increased in allergic patients. However, they did not find the expected increase in GATA-3 expression, suggesting that allergen-induced IL-4 might not be of T-cell origin. We sought to determine whether other cell types were responsible for the increased IL-4 expression in the CD25(+) cell population. METHODS: Comparing six allergic patients and six healthy controls, we analyzed the CD25(+) isolated population from PBMC for the presence of potential IL-4-expressing non-T cells. We also compared spontaneous expression levels of surface markers (CD203c, CD63, CD25, and HLA-DR) on basophils from whole blood of 42 peanut-allergic patients and from 12 non-atopic controls. Expression of these markers was also evaluated following basophil activation in eight peanut-allergic patients selected from the previous cohort. RESULTS: In addition to CD4(+) T cells, a substantial proportion of non-T cells were found in the CD25(+) -isolated cell population: basophils, NK, and NK-T cells with a mean percentage ± s.e.m. of 5.24 ± 0.63%, 6.65 ± 1.01%, and 6.01 ± 1.04%, respectively. The majority of these cells exhibited positive intracytoplasmic staining for IL-4. Expression of CD63 and CD25 was significantly higher in allergic patients compared with controls (p < 0.05). Interestingly, we found a significantly higher proportion of activated basophils expressing HLA-DR, compared with non-activated basophils (p < 0.05). CONCLUSIONS: Our results support the suggested key role of non-T cells secreting IL-4 in food allergy, particularly basophils, which may also play a central role in antigen presentation.

Landscape of epithelial cell subpopulations in the human esophageal squamous cell carcinoma microenvironment.

Aims: We sought to reveal the landscape of epithelial cell subpopulations in the human esophageal squamous cell carcinoma microenvironment and investigate their parts on esophageal squamous carcinoma (ESCC) development. Background: Epithelial cells play an important role in the occurrence and development of ESCC through multiple mechanisms. While the landscape of epithelial cell subpopulations in ESCC, remains unclear. Objective: Exploring the role of epithelial cell subpopulations in ESCC progression. Methods: Seurat R package was used for single-cell RNA sequencing (scRNA-seq) data filtering, dimensionality reduction, clustering and differentially expressed genes analysis. Cellmarker database was adopted for cell cluster annotation. Functional enrichment analysis was carried out by Gene Ontology (GO) analysis. InferCNV package was conducted for copy number variation (CNV) of epithelial cell subpopulations in all chromosomal regions. Pseudotime trajectory analysis was implemented for exploring differentiation trajectory of epithelial cells subgroups during the cancer progression. CellChat analysis was used for probing the interactions between epithelial cells and NK/T cells. cellular experiments were performed using Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR), Wound-Healing Assay and transwell. Results: 11 major cell subpopulations were identified in ESCC and adjunct tissues. Further reclassification of epithelial cells uncovered 4 subpopulations. Enrichment analysis revealed that highly expressed genes in 4 epithelial cell subpopulations were related to cell proliferation, immune response and angiogenesis. CNV analysis found that UBD + epithelial cells and GAS2L3+ epithelial cells had a higher proportion of CNV. Cell differentiation trajectories disclosed that KRT6C+ and GSTA1+ epithelial cells were in an intermediate state of differentiation, while UBD+ and GAS2L3+ epithelial cells are in an end state of differentiation during ESCC progression. Finally, we found that four epithelial cell subpopulations all inhibited NK/T cells through NECTIN2-TIGIT and CLEC2B-KLRB1. Low ATF3 and DDIT3 mRNA expression inhibited ESCC cell migration and invasion. Conclusion: Here, we obtained a through epithelial cell atlas of ESCC at single-cell resolution, explored the role of epithelial cell in ESCC progression, and unveiled immunosuppressive signals to NK/T cells in promoting ESCC. Our findings expand the comprehension of epithelial cells and offer a theoretical guidance for future anti-epithelial cell treatment of ESCC.

Clinical application of cytokine-induced killer (CIK) cell therapy in colorectal cancer: Current strategies and future challenges.

Colorectal cancer (CRC) remains a significant global health burden and is the second leading cause of cancer-related death. Cytokine induced killer (CIK) cell therapy is an immunotherapy which has the potential to meet this need. Clinical trials of CIK cell therapy for the management of CRC have reported improved clinical outcomes. However, production and delivery protocols varied significantly, and many studies were reported only in Chinese language journals. Here we present the most comprehensive review of the clinical CIK cell therapy trials for CRC management to date. We accessed both English and Chinese language clinical studies, and summarise how CIK cell therapy has been implemented, from manufacturing to patient delivery. We discuss current challenges that impede wider adoption of CIK cell therapy in CRC management.

Temporal Immune Profiling in the CSF and Blood of Patients with Aneurysmal Subarachnoid Hemorrhage.

Background: Delayed cerebral ischemia (DCI) is a significant complication of aneurysmal subarachnoid hemorrhage (aSAH). This study profiled immune responses after aSAH and evaluated their association with DCI onset. Methods: Twelve aSAH patients were enrolled. Leukocyte populations and cytokine levels were analyzed in cerebrospinal fluid (CSF) and peripheral blood (PB) on days 3, 5, 7, 10, and 14 post-aSAH. Peripheral blood mononuclear cells (PBMCs) were collected and their cytokine production quantified following stimulation. Results: Mixed-effects models revealed distinct immune cell dynamics in CSF compared to blood. Natural killer T cell frequency increased over time in CSF only, while monocyte/macrophage numbers increased in both CSF and PBMCs. CD4+ HLA II+ T cells increased in circulation. Unstimulated PBMCs showed increased IL-1ß, IL-6, and TNFα production, peaking at 7 days post-aSAH, coinciding with typical DCI onset. Ex vivo stimulation of PBMCs showed that only IL-6 significantly changed over time. In CSF, cytokines peaked 5 days post-injury, preceding immune cell profile alterations. Conclusions: Our findings reveal a time-dependent immune response following aSAH, with distinct within-patient patterns in CSF and PB. The early CSF cytokine peak preceding immune cell changes suggests a potential mechanistic link and identifies the cytokine response as a promising therapeutic target. This cytokine surge may drive immune cell expansion and prime PBMCs for increased inflammatory activity, potentially contributing to DCI risk. Future studies should explore the importance and sources of specific cytokines in driving immune activation. These insights may inform the development of targeted immunomodulatory strategies for preventing or managing DCI in aSAH patients.

NKG2C, HLA-E and their association with psoriasis.

Natural killer (NK) cell activation is regulated by the integration of signals from inhibitory and activating cell surface receptors. Both NKG2A and NKG2C pair with CD94 to form inhibitory and activating receptors specific for the HLA-E-canonical peptide complex. HLA-E is a non-classical MHC class Ib molecule with limited polymorphism. It preferentially binds to and presents leader sequence peptides derived from classical MHC class I molecules. Wilson et al. have identified an association between NKG2C deficiency and psoriasis. They have also discovered an HLA-C-dependent association between HLA-E and psoriasis. Their research highlights the importance of NK cells in the pathophysiology of psoriasis. Herein, we propose two different models to explain the association between NKG2C, HLA-E and psoriasis. In the first model, we hypothesize that NKG2C deficiency and/or HLA-E O1:01 can inhibit the ability of NK cells to regulate autoreactive T cells, predisposing to psoriasis. The second model proposes that HLA-E 01:03 can disrupt the presentation of the psoriasis-inducing self-determinant by HLA-C, thereby protecting against psoriasis.

Exploration of the heterogeneity and interaction of epithelial cells and NK/T-cells in Laryngeal Squamous Cell Carcinoma based on single-cell RNA sequencing data.

OBJECTIVES: We aimed to explore the heterogeneity and differentiation trajectories of epithelial cells and NK/T-cells in Laryngeal Squamous Cell Carcinoma (LSCC). METHODS: We downloaded the GSE150321 data set containing LSCC01 and LSCC02 samples single cell RNA data from Gene Expression Omnibus. The UMAP analysis was performed to identify the cell subpopulations and cell locations of subpopulations. Seurat package was used to analyze the differential expression of genes. The function of differential expression genes was analyzed using DAVID database. The monocle2 package was used to analyze differentiation trajectories. We used the CellChat package to observe the signaling pathways and ligand-receptor pairs for epithelial cells and NK/T-cells. RESULTS: All the LSCC cells were divided into 16 subpopulation that included 7 epithelial cell subsets, 3 T-cell subsets. The function analysis indicated that epithelial cells and NK/T-cells mainly participated in different process, such as cell cycle, immune response, and cell migration. Then, the results of differentiation trajectory indicated that the ability of migration, and the activation of the immune system increases, while the ability of apoptosis, and glucose metabolic process decreases as pseudotime. Migration-related epithelial cells act on all T-cells via the CNTN2-CNTN2 ligand-receptor pair, which suggested that CNTN2 might be an important biomarker for regulating migration of epithelial cells. CONCLUSIONS: Our study characterized the heterogeneity of LSCC, which provided novel insights into LSCC and identified a new mechanism and target for clinical LSCC threapies. EVIDENCE: IV.

Primary Non-Hodgkin's Lymphoma of the Nose: A Rare Case Report.

Extranodal NK/T-cell lymphoma, nasal type (ENKL) is a rare nasal pathology, which in the past was grouped with list of granulomatous diseases. It is an aggressive non-Hodgkin's lymphoma characterized clinically by a non-relenting destruction of the midline structures of the palate and nasal cavity. Despite the malignant clinical nature, tissue diagnosis may be difficult due to extensive tissue necrosis mandating multiple biopsies and has an ominous prognosis, as the average survival rate lying between 6 and 25 months as in large number of Asian studies. This is the case report of a 60-year-old female who presented with (L) nasal obstruction and recurrent episodes of rhinosinusitis for last 8 months, which had been treated with antibiotics, anti-inflammatory drugs and intranasal corticosteroids without success. After performing battery of tests, diagnosed histologically and confirmed by immunohistochemical analysis that the patient had an ENKL, nasal type (AKA angiocentric T-cell lymphoma).

Role of NK T cells in transplantation with particular emphasis on corneal transplantation.

Natural killer T cells (NKT cells) are a unique subset of the immune system that possess characteristics of both an innate and adaptive immune response. This study reviews the reported roles of NKT cells in different solid transplantations such as cardiac, skin, liver, and corneal grafts as well as investigates a novel role of NKT cells in steroid-resistant corneal rejections. It is unknown why there is late corneal graft rejection despite being treated with immunosuppression. Our experimental data suggests NKT cells are playing a crucial part in steroid-resistant late graft rejections. While the pathophysiology of acute rejection is better understood, the process of chronic graft rejection is much less clear. Our data suggests NKT cells as a potential therapeutic target to prevent chronic transplant rejection which needs further investigation.

Case Report: Rhizopus arrhizus Rhino-Orbital-Cerebral Mycosis and Lethal Midline Granuloma: Another Fungal Etiological Agent.

Objective: Both rhino-orbital-cerebral mycosis and lethal midline granuloma (LMG) may result in midline destruction. LMG has now been generally considered as a natural killer/T cell lymphoma, nasal type (ENKTL-NT) with an association of EBV. Fungi have been detected from the diseased tissues now and then but are often considered as lymphoma-associated infections. We previously reported an ENKTL-NT case with Mucor irregularis, which played a causal role in the disease and was involved in the overexpression of Ki67 and CD56 in the mouse experiment. The present study describes a chronic Rhizopus arrhizus infection with immunological parameters that are closely similar to LMG. We aim to explore the relationship of another Mucorales fungus, R. arrhizus, and LMG in a patient and in mice. Methods: Case study and mouse infection modules were designed for our observation. A 35-year-old man with midline face ulcers which was clinically suspected as LMG was selected. Biopsy specimens were sent for lymphoma diagnosis and microbiological detection. The isolated fungus was tested in an ICR mouse model for mycological and histological analyses. Results: Five tissue samples yielded Rhizopus arrhizus. In the pathology, characteristic inflammation, necrosis, and granulation with thin-walled hyphae are observed. Immunohistochemistry showed NK/T cell infiltration (CD3+, CD8+, TIA1+, GZMB+, PRF+, individual CD56+) with hyperplasia (Ki67+) and angioinvasion. The patient recovered completely with amphotericin B. In the murine experiment, R. arrhizus caused angioinvasion with NK/T cell infiltration (CD3+, CD56+, TIA1+, GZMB +, PRF+) with proliferation (Ki67+) and was re-isolated from the infected host. Conclusions: We here describe a mid-face destruction patient, which was diagnosed by the top pathologists in China according to the current criteria of NK/T cell lymphoma, with a negative result for EBV and positive result for R. arrhizus. With a then developed mouse experiment, the R. arrhizus in the diseased lesions was responsible for the NK/T cell infiltration (CD3+, CD8+, CD56+, TIA1+, GZMB+, PRF+), proliferation (Ki67+), and angioinvasion, suggesting another fungal etiological agent for LMG, which could be eradicated with amphotericin B. Limitations: The sample size is not sufficient for statistical analysis. However, our findings are suggestive for the role fungus plays in LMG.

Post-sepsis immunosuppression depends on NKT cell regulation of mTOR/IFN-γ in NK cells.

As treatment of the early, inflammatory phase of sepsis improves, post-sepsis immunosuppression and secondary infection have increased in importance. How early inflammation drives immunosuppression remains unclear. Although IFN-γ typically helps microbial clearance, we found that increased plasma IFN-γ in early clinical sepsis was associated with the later development of secondary Candida infection. Consistent with this observation, we found that exogenous IFN-γ suppressed macrophage phagocytosis of zymosan in vivo, and antibody blockade of IFN-γ after endotoxemia improved survival of secondary candidemia. Transcriptomic analysis of innate lymphocytes during endotoxemia suggested that NKT cells drove IFN-γ production by NK cells via mTORC1. Activation of invariant NKT (iNKT) cells with glycolipid antigen drove immunosuppression. Deletion of iNKT cells in Cd1d-/- mice or inhibition of mTOR by rapamycin reduced immunosuppression and susceptibility to secondary Candida infection. Thus, although rapamycin is typically an immunosuppressive medication, in the context of sepsis, rapamycin has the opposite effect. These results implicated an NKT cell/mTOR/IFN-γ axis in immunosuppression following endotoxemia or sepsis. In summary, in vivo iNKT cells activated mTORC1 in NK cells to produce IFN-γ, which worsened macrophage phagocytosis, clearance of secondary Candida infection, and mortality.

An engineered anti-idiotypic antibody-derived killer peptide (KP) early activates swine inflammatory monocytes, CD3+CD16+ natural killer T cells and CD4+CD8α+ double positive CD8ß+ cytotoxic T lymphocytes associated with TNF-α and IFN-γ secretion.

This study evaluated the early modulation of the phenotype and cytokine secretion in swine immune cells treated with an engineered killer peptide (KP) based on an anti-idiotypic antibody functionally mimicking a yeast killer toxin. The influence of KP on specific immunity was investigated using porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) as ex vivo antigens. Peripheral blood mononuclear cells (PBMC) from healthy pigs were stimulated with KP and with a scramble peptide for 20 min, 1, 4 and 20 h or kept unstimulated. The cells were analyzed using flow cytometry and ELISA. The same time-periods were used for KP pre-incubation/co-incubation to determine the effect on virus-recalled interferon-gamma (IFN-γ) secreting cell (SC) frequencies and single cell IFN-γ productivity using ELISPOT. KP induced an early dose-dependent shift to pro-inflammatory CD172α+CD14+high monocytes and an increase of CD3+CD16+ natural killer (NK) T cells. KP triggered CD8α and CD8ß expression on classical CD4-CD8αß+ cytotoxic T lymphocytes (CTL) and double positive (DP) CD4+CD8α+ Th memory cells (CD4+CD8α+low CD8ß+low). A fraction of DP cells also expressed high levels of CD8α. The two identified DP CD4+CD8α+high CD8ß+low/+high CTL subsets were associated with tumor necrosis factor alpha (TNF-α) and IFN-γ secretion. KP markedly boosted the reactivity and cross-reactivity of PRRSV type-1- and PCV2b-specific IFN-γ SC. The results indicate the efficacy of KP in stimulating Th1-biased immunomodulation and support studies of KP as an immunomodulator or vaccine adjuvant.

Differential Control of iNKT Cell Effector Lineage Differentiation by the Forkhead Box Protein O1 (Foxo1) Transcription Factor.

The invariant NKT (iNKT) cells recognize glycolipid antigens presented by the non-classical MHC like molecule CD1d. They represent an innate T-cell lineage with the ability to rapidly produce a variety of cytokines in response to agonist stimulation to bridge innate and adaptive immunity. In thymus, most iNKT cells complete their maturation and differentiate to multiple effector lineages such as iNKT-1, iNKT-2, and iNKT-17 cells that possess the capability to produce IFNγ, IL-4, and IL-17A, respectively, and play distinct roles in immune responses and diseases. Mechanisms that control iNKT lineage fate decisions are still not well understood. Evidence has revealed critical roles of Foxo1 of the forkhead box O1 subfamily of transcription factors in the immune system. However, its role in iNKT cells has been unknown. In this report, we demonstrate that deletion of Foxo1 causes severe decreases of iNKT cell total numbers due to impairment of late but not early iNKT cell development. Deficiency of Foxo1 results in decreases of iNKT-1 but increases of iNKT-17 cells. Our data reveal that Foxo1 controls iNKT effector lineage fate decision by promoting iNKT-1 but suppressing iNKT-17 lineages.

Granulysin, a novel marker for extranodal NK/T cell lymphoma, nasal type.

Granulysin is a cytolytic protein expressed in cytotoxic T and natural killer (NK) cells. Abnormal serum levels of granulysin in lymphomas with NK and cytotoxic phenotype have been shown to correlate with tumour progression. In this study, we investigated the expression pattern of granulysin in routine sections of normal and reactive lymphoid tissues as well as in a large series of lymphomas. In normal tissues, granulysin labelled a small population of cells that double immunostaining revealed to belong to the pool of cytotoxic T/NK cells. Among lymphoid neoplasms, the highest expression of granulysin (71%) was found in extranodal NK/T cell lymphomas of nasal type (ENKTL). To note is that 29% of ENKTLs, which were negative for one or more of classical cytotoxic markers strongly expressed granulysin. Furthermore, expression of granulysin was observed in rare cases of T cell lymphomas with a cytotoxic phenotype (i.e. ALK-negative anaplastic large cell lymphoma (26%), enteropathy-associated T cell lymphoma (12%) and peripheral T cell lymphoma, NOS (4%)). None of the investigated non-Hodgkin B cell lymphomas, Hodgkin lymphoma and plasma cell myeloma were granulysin positive. The results suggest granulysin as a novel marker for a subset of cytotoxic NK cell derived malignancies and its usefulness is highlighted in those ENKTLs that lack expression of other cytotoxic markers but retain granulysin expression.

Induction of antiinflammatory purinergic signaling in activated human iNKT cells.

Invariant natural killer T (iNKT) cells are activated at sites of local tissue injury, or globally during vaso-occlusive episodes of sickle cell disease (SCD). Tissue damage stimulates production of CD1d-restricted lipid antigens that activate iNKT cells to produce Th1- and Th2-type cytokines. Here, we show that circulating iNKT cells in SCD patients express elevated levels of the ectonucleoside triphosphate diphosphosphohydrolase, CD39, as well the adenosine A2A receptor (A2AR). We also investigated the effects of stimulating cultured human iNKT cells on the expression of genes involved in the regulation of purinergic signaling. iNKT cell stimulation caused induction of ADORA2A, P2RX7, CD38, CD39, ENPP1, CD73, PANX1, and ENT1. Transcription of ADA, which degrades adenosine, was reduced. Induction of CD39 mRNA was associated with increased ecto-ATPase activity on iNKT cells that was blocked by POM1. Exposure of iNKT cells to A2AR agonists during stimulation reduced production of IFN-γ and enhanced production of IL-13 and CD39. Based on these findings, we define "purinergic Th2-type cytokine bias" as an antiinflammatory purinergic response to iNKT cell stimulation resulting from changes in the transcription of several genes involved in purine release, extracellular metabolism, and signaling.

Age-related M1/M2 phenotype changes in circulating monocytes from healthy/unhealthy individuals.

Macrophage polarization is a candidate biomarker of disease-related inflammatory status, but its modulation during aging has not been investigated. To do this, the M1/M2 profile was assessed by CD80/CD163 gating in classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14lowCD16+) monocytes from 31 healthy subjects (CTRs) of different ages. Cytofluorimetric analysis showed a significantly different CD80/CD163 distribution in the three subsets, as more than 80% of classical and intermediate monocytes were CD80+CD163+, whereas most non-classical monocytes were CD80-CD163- and CD163+. Non-classical CD163+ monocytes were significantly higher whereas classical CD163+ and CD80-CD163- monocytes significantly lower in older than younger CTRs (cut-off, 65 years), suggesting different age-related trends for M2 subsets. To establish whether an M1/M2 imbalance could be associated with disease, 21 patients with acute myocardial infarction (AMI) were compared with older CTRs. The AMI patients showed a significantly decreased proportion of CD163+CD80+ and an increased proportion of CD163+ and CD163-CD80- cells among classical monocytes, opposite trends to those observed in healthy aging. Moreover, a significantly greater proportion of intermediate and non-classical CD80+ monocytes suggested a shift to a pro-inflammatory phenotype. Overall, CD163/CD80 cytofluorimetric characterization of circulating monocytes provides additional information about their polarization and could be an innovative tool to monitor aging.

Exploration of the heterogeneity and interaction of epithelial cells and NK/T-cells in Laryngeal Squamous Cell Carcinoma based on single-cell RNA sequencing data

Abstract Objectives We aimed to explore the heterogeneity and differentiation trajectories of epithelial cells and NK/T-cells in Laryngeal Squamous Cell Carcinoma (LSCC). Methods We downloaded the GSE150321 data set containing LSCC01 and LSCC02 samples single cell RNA data from Gene Expression Omnibus. The UMAP analysis was performed to identify the cell subpopulations and cell locations of subpopulations. Seurat package was used to analyze the differential expression of genes. The function of differential expression genes was analyzed using DAVID database. The monocle2 package was used to analyze differentiation trajectories. We used the CellChat package to observe the signaling pathways and ligand-receptor pairs for epithelial cells and NK/T-cells. Results All the LSCC cells were divided into 16 subpopulation that included 7 epithelial cell subsets, 3 T-cell subsets. The function analysis indicated that epithelial cells and NK/T-cells mainly participated in different process, such as cell cycle, immune response, and cell migration. Then, the results of differentiation trajectory indicated that the ability of migration, and the activation of the immune system increases, while the ability of apoptosis, and glucose metabolic process decreases as pseudotime. Migration-related epithelial cells act on all T-cells via the CNTN2-CNTN2 ligand-receptor pair, which suggested that CNTN2 might be an important biomarker for regulating migration of epithelial cells. Conclusions Our study characterized the heterogeneity of LSCC, which provided novel insights into LSCC and identified a new mechanism and target for clinical LSCC threapies. Evidence IV.

Frequency and perforin expression of different lymphocyte subpopulations in patients with lower limb fracture and thoracic injury.

INTRODUCTION: Trauma with multiple injuries is associated with a high risk of complications, which may be related to excessive stimulation of inflammatory and anti-inflammatory responses. Although the effects of polytrauma on the immune response have been well established at the cellular and molecular levels, there is little information about the changes in the cytolytic potential of immunocompetent cells, including expression of cytotoxic molecules such as perforin. Therefore, the objective of the present study was to analyse and compare differences in the frequency and perforin expression of leukocyte subpopulations in the peripheral blood of patients with lower limb fracture, thoracic injury, and simultaneous lower limb fracture and thoracic injury. PATIENTS AND METHODS: Forty-five patients with trauma injury (15 patients with lower limb injury, 15 patients with thoracic injury, and 15 patients with simultaneous lower limb and thoracic injury) were included in the study. Peripheral blood of 15 sex- and age-matched healthy volunteers served as the control group. Peripheral blood samples were taken from all subjects included in the study and peripheral blood mononuclear cells were isolated by gradient centrifugation. The frequency of T lymphocytes, natural killer (NK) and NK T cells, and their subsets, as well as their perforin expression levels were simultaneously detected and analysed by flow cytometry. RESULTS: There was a statistically significant decrease in the frequency of T lymphocytes, NK and NK T cells as well as perforin expression in the patients with simultaneous lower limb and thoracic injury compared with the other two groups, with a predominantly marked decrease in NK and NK T cells. CONCLUSION: The decrease in the frequency and cytotoxic potential of peripheral blood lymphocytes is related to the severity of trauma injury, which can explain the underlying mechanism contributing to complication occurrence.