Invasive urinary bladder cancer shows high recurrence rates after cystectomy even with apparent complete downstaging at cystectomy. Exosomes are nano-sized vesicles important in cell-cell ...communication, which have been hypothesized to contribute to cancer dissemination and recurrence. The aim of this study was to investigate if pro-carcinogenic exosomes could be detected in urine from histologically downstaged bladder cancer patients. 13 Patients were included in this study. Paired ureter and urine samples from nine patients underwent mass spectrometry, while samples from the remaining patients were used for exosome characterization. At cystectomy, exosomes were isolated from bladder and ureter urine, whereafter quantitative proteome profiling was performed. Urinary exosomes clustered based on whether they came from the bladder, with tumour contact, or the ureters, without tumour contact, even though all came from completely downstaged patients. Proteins overexpressed in exosomes derived from bladder urine contained several oncogenes and were mainly associated with tumour metabolism pathways. Although patients were histologically tumour-free at cystectomy, the bladder urine contained exosomes with a carcinogenic metabolic profile. This suggests a continuous release of exosomes from the bladder, which may promote recurrence at distant sites through metabolic rewiring, even after apparent complete downstaging. These exosomes, coming from either undetected cancer cells or partly transformed cells, are likely to increase the risk of metastasis and encourages cystectomy even in completely downstaged patients.
Recurrence in oral squamous cell carcinoma (OSCC) significantly reduces overall survival. Improved understanding of the host's immune status in head and neck cancer may facilitate identification of ...patients at higher risk of recurrence and improve patients' selection for ongoing clinical trials assessing the effectiveness of immune checkpoint inhibitors (CPI). We aimed to investigate Sentinel Node-derived T-cells and their impact on survival. We enrolled prospectively 28 OSCC patients treated at Karolinska University Hospital, Stockholm, Sweden with primary tumour excision and elective neck dissection. On top of the standard treatment, the enrolled patients underwent sentinel node procedure. T cells derived from Sentinel nodes, non-sentinel nodes, primary tumour and PBMC were analyzed in flow cytometry. Patients who developed recurrence proved to have significantly lower level of CD4+ CD69+ in their sentinel node (31.38 ± 6.019% vs. 43.44 ± 15.33%, p = 0.0103) and significantly higher level of CD8+ CD HLA-DR+ (38.95 ± 9.479% vs. 24.58 ± 11.36%, p = 0.0116) compared to disease-free individuals. Survival analysis of studied population revealed that patients with low proportion of CD4+ CD69+ had significantly decreased disease-free survival (DFS) of 19.7 months (95% CI 12.6-26.9) compared with 42.6 months (95% CI 40.1-45.1) in those with high CD4+ CD69+ proportion in their Sentinel Nodes (log-rank test, p = 0.033). Our findings demonstrate that characterization of T-cell activation in Sentinel Node serves as a complementary prognostic marker. Flow cytometry of Sentinel Node may be useful in both patients' surveillance and selection for ongoing CPI clinical trials in head and neck cancer.
What's known on the subject? and What does the study add?
This is the first study examining FOXP3 expression in invasive urothelial urinary bladder cancer and in their tumour‐infiltrating lymphocytes ...(TILs). The relation of their respective immunohistological expression to survival adds new knowledge in the fields of tumour immunology and prognostic markers.
OBJECTIVE
• To investigate the possible impact of FOXP3 expression in T‐cells, as well as in tumour cells, on long‐term survival in patients with urinary bladder cancer (UBC) invading muscle.
PATIENTS AND METHODS
• In a retrospective study, tumour specimens from 37 patients cystectomized for T1–T4 UBC during 1999–2002 at the Karolinska University Hospital were examined by immunohistochemistry for tumour expression and/or infiltration of immune cells expressing FOXP3 as well as CD3.
• The results obtained were correlated with clinicopathological parameters, where the primary and secondary outcomes investigated were overall survival and progression‐free survival, respectively.
RESULTS
• Infiltration of CD3+ and FOXP3+ lymphocytes (≥3 cells per high‐power field) were both correlated with better survival, and this relationship persisted throughout the whole study period (all P < 0.05).
• Patients with FOXP3+ tumour cells had decreased long‐term survival compared to those patients with FOXP3− tumours (P < 0.05).
• Despite a limited amount of patient material, the results of the present study indicate that FOXP3 expression, in both lymphocytes and tumour cells, is an important prognostic factor in UBC.
CONCLUSIONS
• FOXP3 expression in UBC cells is associated with decreased long‐term survival and thus may be a novel negative prognostic factor in UBC invading muscle.
• By contrast, the presence of FOXP3+ tumour‐infiltrating lymphocytes was correlated with a positive prognosis. Because FOXP3 is up‐regulated upon activation in human T‐cells, FOXP3 may serve more as an activation marker than as a regulatory T‐cell indicator in this case.
• These results support the need for larger prospective studies aiming to confirm the results obtained and to examine the underlying mechanisms in detail.
Hemodialysis (HD) patients have an increased risk of acquiring infections due to many health care contacts and may, in addition, have a suboptimal response to vaccination and a high mortality from ...Covid-19 infection. In 50 HD patients (mean age 69.4 years, 62% men) administration of SARS-CoV-2BNT162b2 mRNA vaccine began in Dec 2020 and the immune response was evaluated 7-15 weeks after the last dose. Levels of Covid-19 (SARS-CoV-2) IgG antibody against the nucleocapsid antigen (anti-N) and the Spike antigen (anti-S) and T-cell reactivity testing against the Spike protein using ELISPOT technology were evaluated. Out of 50 patients, anti-S IgG antibodies indicating a vaccine effect or previous Covid-19 infection, were detected in 37 (74%), 5 (10%) had a borderline response and 8 (16%) were negative after two doses of vaccine. T-cell responses were detected in 29 (58%). Of the 37 patients with anti-S antibodies, 25 (68%) had a measurable T-cell response. 2 (40%) out of 5 patients with borderline anti-S and 2 (25%) without anti-S had a concomitant T-cell response. Twenty-seven (54%) had both an antibody and T-cell response. IgG antibodies to anti-N indicating a previous Covid-19 disease were detected in 7 (14%) patients. Most HD patients develop a B- and/or T-cell response after vaccination against Covid-19 but approx. 20% had a limited immunological response. T-cell reactivity against Covid-19 was only present in a few of the anti-S antibody negative patients.
Background MicroRNAs (miRNAs) are short noncoding RNAs that suppress gene expression at the posttranscriptional level. Atopic dermatitis is a common chronic inflammatory skin disease characterized by ...the presence of activated T cells within the skin. Objective We sought to explore the role of miRNAs in the pathogenesis of atopic dermatitis. Methods Global miRNA expression in healthy and lesional skin of patients with atopic dermatitis was compared by using TaqMan MicroRNA Low Density Arrays. miR-155 expression in tissues and cells was quantified by means of quantitative real-time PCR. The cellular localization of miR-155 was analyzed by means of in situ hybridization. The regulation of cytotoxic T lymphocyte–associated antigen (CTLA-4) by miR-155 was investigated by using luciferase reporter assays and flow cytometry. CTLA-4 expression and functional assays were performed on TH cells overexpressing miR-155. Results miR-155 was one of the highest-ranked upregulated miRNAs in patients with atopic dermatitis. In the skin miR-155 was predominantly expressed in infiltrating immune cells. miR-155 was upregulated during T-cell differentiation/activation and was markedly induced by T-cell activators in PBMCs in vitro and by superantigens and allergens in the skin in vivo . CTLA-4, an important negative regulator of T-cell activation, was identified as a direct target of miR-155. Overexpression of miR-155 in TH cells resulted in decreased CTLA-4 levels accompanied by an increased proliferative response. Conclusion miR-155 is significantly overexpressed in patients with atopic dermatitis and might contribute to chronic skin inflammation by increasing the proliferative response of TH cells through the downregulation of CTLA-4.
A re-examination of former concepts is required to meet today's medical challenges in allergic rhinitis. Previously, neutrophils have been treated as a relatively homogenous cell population found in ...the nose both when the patient is suffering at the height of the allergic season as well as when the patient report no symptoms. However, new data indicates that neutrophils can be divided into different subsets with diverse roles in inflammation. We showed increased levels of neutrophils in peripheral blood, nasal biopsies and nasal lavage fluid (NAL) from allergic patients during the pollen season compared to healthy controls. A closer examination revealed that the activated subset of neutrophils, CD16
CD62L
, outweighed the normal form CD16
CD62L
in nasal tissue among these patients. This skewed distribution was not seen in controls. The normal subset prevailed in peripheral blood from patients as well as controls, whereas CD16
CD62L
and CD16
CD62L
subsets, the latter considered "end state" neutrophils before apoptosis, were elevated in NAL. Functional in vitro experiments revealed that activated neutrophils exhibit a T cell priming capacity and an ability to enhance eosinophil migration. Activated neutrophils may thus contribute to allergic inflammation seen in allergic rhinitis by priming T cells and attracting eosinophils.
Ro52/Trim21 is targeted as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome. Polymorphisms in the Ro52 gene have been linked to these autoimmune conditions, but the molecular ...mechanism by which Ro52 may promote development of systemic autoimmune diseases has not been explored. To address this issue, we generated Ro52-null mice (Ro52(-/-)), which appear phenotypically normal if left unmanipulated. However, Ro52(-/-) mice develop severe dermatitis extending from the site of tissue injury induced by ear tags. The affected mice further develop several signs of systemic lupus with hypergammaglobulinemia, autoantibodies to DNA, proteinuria, and kidney pathology. Ro52, which was recently identified as an E3 ligase, mediates ubiquitination of several members of the interferon regulatory factor (IRF) family, and the Ro52-deficient mice have an enhanced production of proinflammatory cytokines that are regulated by the IRF transcription factors, including cytokines involved in the Th17 pathway (interleukin IL 6, IL-12/IL-23p40, and IL-17). Loss of IL-23/IL-17 by genetic deletion of IL-23/p19 in the Ro52(-/-) mice conferred protection from skin disease and systemic autoimmunity. These data reveal that the lupus-associated Ro52 protein is an important negative regulator of proinflammatory cytokine production, and they provide a mechanism by which a defective Ro52 function can lead to tissue inflammation and systemic autoimmunity through the IL-23-Th17 pathway.
Background
An increase of regulatory T cells, defined as CD25
high
- and/or FOXP3
+
-expressing CD4
+
T cells, within tumors has been reported in several studies. Tregs promote tumor growth by ...modulating the antitumor immune response, mainly through inhibition of T-cell-mediated tumor cell killing: this has been suggested to be dependent on IL-10 and/or TGF-β. In stomach cancer, the mechanisms behind the accumulation of Tregs in tumor tissue has not been fully elucidated, and neither has Treg gene expression in situ.
Materials and methods
Stomach tissue from gastric cancer patients undergoing gastric resection was analyzed using flow cytometry and cell sorting, followed by RT-PCR.
Results
We observed that stomach CD4
+
FOXP3
+
T cells proliferated to a higher degree than CD4
+
FOXP3
−
T cells, which may contribute to Treg accumulation in the mucosa. By analyzing DNA methylation, we demonstrated that both proliferating and nonproliferating FOXP3
+
T cells exhibited complete demethylation of the
FOXP3
gene, indicating a stable FOXP3 expression in both cell populations. Furthermore, analysis of T-cell populations isolated directly from the tumor and tumor-free mucosa demonstrated that CD4
+
CD25
high
T cells have a higher IL-10/IFN-γ gene expression ratio but express lower levels of TGF-β than CD4
+
CD25
low/−
T cells.
Conclusion
We demonstrate strong proliferation among regulatory CD4
+
FOXP3
+
CD25
high
T cells in the gastric cancer mucosa. These local Treg express a suppressive cytokine profile characterized by high IL-10 and low TGF-β and IFN-γ production.
Hypoxic ischemia (HI) is an important cause of neonatal brain injury and subsequent inflammation affects neurological outcome. In this study we performed investigations of systemic and local ...activation states of inflammatory cells from innate and adaptive immunity at different time points after neonatal HI brain injury in mice.
We developed a multiplex flow cytometry based method combined with immunohistochemistry to investigate cellular immune responses in the brain 24 h to 7 months after HI brain injury. In addition, functional studies of ex vivo splenocytes after cerebral hypoxic ischemia were performed. Both central and peripheral activation of CD11b(+) and CD11c(+) antigen presenting cells were seen with expression of the costimulatory molecule CD86 and MHC-II, indicating active antigen presentation in the damaged hemisphere and in the spleen. After one week, naïve CD45rb(+) T-lymphocytes were demonstrated in the damaged brain hemisphere. In a second phase after three months, pronounced activation of CD45rb(-) T-lymphocytes expressing CD69 and CD25 was seen in the damaged hemisphere. Brain homogenate induced proliferation in splenocytes after HI but not in controls.
Our findings demonstrate activation of both local and systemic immune responses months after hypoxic ischemic neonatal brain injury. The long term immune activation observed is of general importance for future studies of the inflammatory response after brain injury as most previous studies have focused on the first few weeks after damage, while the effects of the late inflammation phase may be missed. Furthermore, the self-reactive component raises the question if there is a correlation with development of autoimmune brain disease later in life.
Naturally occurring thymus derived regulatory T cells (Tregs) are central in the maintenance of self-tolerance. The transcription factor FOXP3 is crucial for the suppressive activity of Tregs and is ...considered the most specific marker for this population. However, human non regulatory T cells upregulate FOXP3 transiently upon activation which calls for other means to identify the Treg population. Since epigenetic mechanisms are involved in the establishment of stable gene expression patterns during cell differentiation, we hypothesized that the methylation profile of the FOXP3 promoter would allow the distinction of truly committed Tregs.
Human CD4(+)CD25(hi) Tregs displayed a demethylated FOXP3 promoter (1.4%+/-0.95% SEM methylated) in contrast to CD4(+)CD25(lo) T cells which were partially methylated (27.9%+/-7.1%). Furthermore, stimulated CD4(+)CD25(lo) T cells transiently expressed FOXP3 but remained partially methylated, suggesting promoter methylation as a mechanism for regulation of stable FOXP3 expression and Treg commitment. In addition, transient FOXP3 expressing cells exhibited suppressive abilities that correlate to the methylation status of the FOXP3 promoter. As an alternative to bisulphite sequencing, we present a restriction enzyme based screening method for the identification of committed Tregs and apply this method to evaluate the effect of various culturing conditions. We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-beta and/or IL-10 does not induce any additional change in methylation level.
The unique FOXP3 promoter methylation profile in Tregs suggests that a demethylated pattern is a prerequisite for stable FOXP3 expression and suppressive phenotype. Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy. In these settings there is a need to distinguish true Tregs from transiently FOXP3(+) activated T cells. The screening method we present allows this distinction and enables the identification of cells suitable for in vitro expansions and clinical use.
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