Transcriptional profiling demonstrates altered characteristics of CD8+ cytotoxic T-cells and regulatory T-cells in TP53-mutated acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) patients have limited effect from T-cell-based therapies, such as PD-1 and CTLA-4 blockade. However, recent data indicate that AML patients with TP53 mutation have higher immune infiltration and other immunomodulatory therapies could thus potentially be effective. Here, we performed the transcriptional analysis of distinct T-cell subpopulations from TP53-mutated AML to identify gene expression signatures suggestive of altered functional properties. METHODS: CD8+ cytotoxic T lymphocytes (CTLs), conventional helper T cells (Th), and regulatory T cells (Tregs) were sorted from peripheral blood of AML patients with TP53 mutation (n = 5) and healthy donors (n = 3), using FACS, and the different subpopulations were subsequently subjected to RNA-sequencing. Differentially expressed genes were identified and gene set enrichment analysis (GSEA) was performed to outline altered pathways and exhaustion status. Also, expression levels for a set of genes encoding established and emerging immuno-oncological targets were defined. RESULTS: The results showed altered transcriptional profiles for each of the T-cell subpopulations from TP53-mutated AML as compared to control subjects. IFN-α and IFN-γ signaling were stronger in TP53-mutated AML for both CTLs and Tregs. Furthermore, in TP53-mutated AML as compared to healthy controls, Tregs showed gene expression signatures suggestive of metabolic adaptation to their environment, whereas CTLs exhibited features of exhaustion/dysfunction with a stronger expression of TIM3 as well as enrichment of a gene set related to exhaustion. CONCLUSIONS: The results provide insights on mechanisms underlying the inadequate immune response to leukemic cells in TP53-mutated AML and open up for further exploration toward novel treatment regimens for these patients.

Pattern recognition receptor expression and maturation profile of dendritic cell subtypes in human tonsils and lymph nodes.

Dendritic cells (DCs) with capacity of antigen cross-presentation are of key interest for immunotherapy against cancer as they can induce antigen-specific cytotoxic T lymphocyte (CTL) responses. This study describes frequencies of DC subtypes in human tonsils and lymph nodes, and phenotypic aspects that may be targeted by adjuvant measures. From human tonsils and neck lymph nodes, DCs were identified through flow cytometry, and subsets of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were investigated. Maturity status was assessed and surface receptors with CTL-promoting potentials were studied. CD123+ pDCs as well as CD1c+, CD141+, and CD1c-CD141- mDCs were detected in tonsils and lymph nodes. Both sites featured a similar presence of DC subsets, with CD123+ pDC being dominant and CD141+ mDCs least frequent. Based on CD80/CD86 expression, all DC subtypes featured a low degree of maturation. Expression of pattern recognition receptors (PRRs) CD206, CD207, DC-SIGN, TLR2, and TLR4, as well as the chemokine receptor XCR1, indicated DC subset-specific receptor profiles. We conclude that tonsils and lymph nodes share common features in terms of DC subset frequency and maturation as well as PRR and XCR1 expression pattern. Our work suggests that both sites may be considered for vaccine deposition in DC-mediated immunotherapy.

Dendritic cell subpopulations in nasopharyngeal cancer.

Nasopharyngeal cancer (NPC) is associated with Epstein-Barr virus (EBV) and EBV antigen may be utilized for therapeutic purposes, including targeting of dendritic cells (DCs). Although DCs may be present in NPC, the information is limited and not up-to-date with current knowledge on DC subsets. In the present study, biopsies from untreated NPC were obtained and subjected to multicolor flow-cytometry focusing on DC subtype markers: CD123 for plasmacytoid DCs (pDCs); and CD1c and CD141 for myeloid DCs (mDCs). Furthermore, subset-specific expression of the C-lectin receptor (CLR) CD207 (also termed langerin) was assessed. pDCs and mDCs were detected in the NPC lesions, contributing to a frequency mean average of 0.78% of CD45+ leukocytes in situ. Different subpopulations, previously not described in NPC, were observed, including: CD123+ pDCs; CD1c+ mDCs; CD141+ mDCs; and CD1c-CD141- mDCs. A high frequency of CD1c+ mDCs expressing CD207 was observed, compared with other subsets. In conclusion, different DC subsets are present in NPC lesions. The CLR CD207, a selective endocytic marker on CD1c+ mDCs, may be targeted for therapeutic purposes to facilitate cross-presentation of antigens and aid cell-mediated antitumor effects.

Profiling dendritic cell subsets in head and neck squamous cell tonsillar cancer and benign tonsils.

Dendritic cells (DCs) have a key role in orchestrating immune responses and are considered important targets for immunotherapy against cancer. In order to develop effective cancer vaccines, detailed knowledge of the micromilieu in cancer lesions is warranted. In this study, flow cytometry and human transcriptome arrays were used to characterize subsets of DCs in head and neck squamous cell tonsillar cancer and compare them to their counterparts in benign tonsils to evaluate subset-selective biomarkers associated with tonsillar cancer. We describe, for the first time, four subsets of DCs in tonsillar cancer: CD123+ plasmacytoid DCs (pDC), CD1c+, CD141+, and CD1c-CD141- myeloid DCs (mDC). An increased frequency of DCs and an elevated mDC/pDC ratio were shown in malignant compared to benign tonsillar tissue. The microarray data demonstrates characteristics specific for tonsil cancer DC subsets, including expression of immunosuppressive molecules and lower expression levels of genes involved in development of effector immune responses in DCs in malignant tonsillar tissue, compared to their counterparts in benign tonsillar tissue. Finally, we present target candidates selectively expressed by different DC subsets in malignant tonsils and confirm expression of CD206/MRC1 and CD207/Langerin on CD1c+ DCs at protein level. This study descibes DC characteristics in the context of head and neck cancer and add valuable steps towards future DC-based therapies against tonsillar cancer.

Clinical efficacy of a topical lactic acid bacterial microbiome in chronic rhinosinusitis: A randomized controlled trial.

Objective: A locally disturbed commensal microbiome might be an etiological factor in chronic rhinosinusitis (CRS) in general and in CRS without nasal polyps (CRSsNP) in particular. Lactic acid bacteria (LAB) have been suggested to restore commensal microbiomes. A honeybee LAB microbiome consisting of various lactobacilli and bifidobacteria have been found potent against CRS pathogens in vitro. Recently, we examined effects of single nasal administrations of this microbiome in healthy subjects and found it inert. In this study, we examined effects of repeated such administrations in patients with CRSsNP. Study Design: The study was of a randomized, double-blinded, crossover, and sham-controlled design. Methods: Twenty patients received 2 weeks' treatment administered using a nasal spray-device. The subjects were monitored with regard to symptoms (SNOT-22 questionnaire, i.e., the primary efficacy variable), changes to their microbiome, and inflammatory products (IL-6, IL-8, TNF-, IL-8,a, and MPO) in nasal lavage fluids. Results: Neither symptom scores, microbiological explorations, nor levels of inflammatory products in nasal lavage fluids were affected by LAB (c.f. sham). Conclusion: Two weeks' nasal administration of a honeybee LAB microbiome to patients with CRSsNP is well tolerated but affects neither symptom severity nor the microbiological flora/local inflammatory activity. Level of Evidence: 1b.

Interleukin-1 beta, interferon-gamma, and tumor necrosis factor-alpha gene expression in peripheral blood mononuclear cells of patients with coronary artery disease.

BACKGROUND: Several inflammatory mediators have been proposed to contribute to the pathogenesis of atherosclerosis. The aim of this study was to evaluate the quantitative expression of pro-inflammatory cytokines in un-stimulated peripheral blood mononuclear cell of patients with coronary artery disease (CAD). METHODS: Interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha, and interferon-gamma (IFN-γ) gene expression were evaluated in angiography confirmed patients with and without CAD in a case-control study using quantitative real-time polymerase chain reaction. RESULTS: A significant increase (P = 0.030) in IL-1ß gene expression was found in patients with CAD [median interquartile range (IQR) = 4.890 (6.084)] compared to patients without CAD [median (IQR) = 1.792 (3.172)]. Despite the increase in IFN-γ gene expression in patients with CAD [median (IQR) = 1.298 (3.896)] versus patients without CAD [median (IQR) = 0.841 (2.79)], there was not statistically significant difference (P = 0.990). CONCLUSION: Our results provide evidence for possible association between IL-1ß and development of atherosclerosis as a crucial cytokine that induce a network of signaling pathways. This finding if proved in future would suggest IL-1ß as a potent therapeutic target in CAD.

eNOS Gene Variant in Patients with Coronary Artery Disease.

Subject & Aim. Endothelial nitric oxide synthase (eNOS) is one of the most important candidate genes in CAD. A functional polymorphism within eNOS gene is a 27 bp VNTR on its intron 4 which has been shown to be associated with various diseases. In this study we investigated eNOS VNTR polymorphism in addition to eNOS gene expression profile in patients with CAD. Material and Methods. The study comprised patients with angiographically confirmed CAD (CAD(+)) and individuals with normal coronary as CAD(-). eNOS VNTR polymorphism frequencies were determined in both groups. In addition eNOS gene expression profile was examined using a quantitative real-time PCR. Results. We have found that aa genotype was significantly increasing the risk of CAD in our patients (aa versus ab + bb, P = 0.02, OR = 3.5; 95% CI: = 0.98 to 16.2). The differences in eNOS expression were not significant between patients and normal group; however in CAD(+) patients eNOS expression was higher than the expression level of patients carrying other genotypes (P = 0.16). Conclusion. We have observed that eNOS gene polymorphism was associated with CAD in angiography-confirmed patients. However, the difference in eNOS gene expression was not statistically significant between patients and control which might be due to the contribution of other confounding factors which require further investigations.

IL-23 gene expression in PBMCs of patients with coronary artery disease.

OBJECTIVE: Both adaptive and innate immune systems are involved in coronary artery disease (CAD). The aim of this study was to evaluate TH17 cytokines expression profiles in un-stimulated peripheral blood lymphocytes (PBMCs) of patients with coronary artery disease. METHODS: Expression profiles of IL-17, IL-23, and TGF-ß1 were determined in individuals with and without CAD using Real-time PCR. RESULTS: A significant decrease in IL-23 gene expression in un-stimulated PBMCs of patients with CAD compared to those without CAD was found (p=0.003, OR=0.045, 95% CI: 0.006-0.355). CONCLUSION: Our data reinforce the potential role of the IL-23 as a critical regulatory molecule that bridges the innate and adaptive arms of the immune system in the complex mechanisms associated with the development of atherosclerosis.

TGF-ß and IL-23 gene expression in unstimulated PBMCs of patients with diabetes.

The protective effects of TGF-ß have been documented in various autoimmune diseases, mostly in organ-specific autoimmunity including type 1 diabetes mellitus (T1DM). However, TGF-ß also plays a role as a pro-inflammatory mediator by induction of Th17 cytokine production. IL-23 also plays a key role in differentiation of Th17 cells, which are implicated in pathogenesis of autoimmune conditions including T1DM. The aim of this study was to investigate and compare the difference in the level of TGF-ß1 and IL-23 gene expression in unstimulated peripheral blood mononuclear cells (PBMCs) of patients with different forms of diabetes compared with normal healthy controls subjects. Patients with T1DM were grouped as early-onset T1DM (N = 20) with age at diagnosis <18 years and late-onset T1DM (N = 20) with the age at onset >18 years. Patients with T2DM (N = 20) and normal healthy controls (N = 20) were recruited from the same area. TGF-ß1 and IL-23 gene expression in fresh unstimulated PBMCs was determined in each group using quantitative real-time PCR. The results confirmed that a significant difference in TGF-ß1 and IL-23 gene expression was observed in both forms of juvenile-onset T1DM and adult-onset T1DM compared to the controls and T2DM patients. There was no significant difference for TGF-ß gene expression in patients with T2DM and controls. We therefore conclude that our results support the previous data on TGF-ß gene down-regulation in T1DM. Also up-regulation of IL-23 has been observed in T1DM whilst it was down-regulated in T2DM. We also found no significant difference between juvenile-onset and adult-onset T1DM indicating same mechanism might be involved in the pathogenesis of both types. More studies on different cytokines in Th17 pathways are required to further confirm our finding.