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Background: Although therapeutic human papillomavirus vaccines could offer a noninvasive treatment for patients with cervical intraepithelial neoplasia, none has been clinically implemented. Oral administration of the therapeutic human papillomavirus vaccine IGMKK16E7 results in the histological regression of human papillomavirus 16-positive cervical intraepithelial neoplasia 2/3 to normal (complete response). We investigated biomarkers that could predict complete response after oral administration of IGMKK16E7., Methods: Forty-two patients administered high-dose oral IGMKK16E7 in a phase I/II trial were included. Cervix-exfoliated cells were collected before vaccine administration. Gene expression of CD4, CD8, FOXP3, programmed cell death 1 protein, CTLA4, CD103, CD28, CD80, CD86, and programmed cell death 1 ligand 1 in the cells was measured by quantitative reverse transcriptase-polymerase chain reaction. Receiver operating characteristic curve analysis and Mann-Whitney tests were used to explore potential biomarkers. Pearson correlation coefficient analysis was used to correlate gene expression profiles with clinical outcome., Results: The only predictive biomarker of vaccine response for which receiver operating characteristic curve analysis showed significant diagnostic performance with histological complete response was CD86 (area under the curve = 0.71, 95% confidence interval = 0.53 to 0.88, P = .020). Patients with complete response had significantly lower CD86 expression (CD86-low) than patients with no complete response (P = .035). The complete response rates for CD86-low and CD86-high patients were 50% and 19%, respectively, and CD86-low patients had a significantly higher complete response rate (P = .047). Compared with all patients, the CD86-low group had a 1.5-fold increase in the complete response rate. Gene expression of CD86 and CTLA4 showed the strongest positive correlation with clinical outcomes in the incomplete response group (P < .001)., Conclusion: Low expression of CD86 in exfoliated cervical cells can be used as a pretreatment biomarker to predict histological complete response after IGMKK16E7 administration., (© The Author(s) 2024. Published by Oxford University Press.)

Aims: Angioimmunoblastic T cell lymphoma (AITL) is a T cell lymphoma with aberrant immune activity. It is characterised by inflammatory and immune reactions. However, the impact of regulatory T (Treg) cells on AITL remains unclear., Methods: We retrospectively collected 46 AITL cases and performed immunohistochemical analysis of forkhead box P3 (FOXP3) expression. The number of immunostained FOXP3 cells was determined using a digital pathology system with whole-slide imaging. The average number of FOXP3+ cells per high-power field (HPF) was determined by randomly counting 20 HPFs. AITL cases were categorised into high-expression and low-expression groups based on the median count of FOXP3+ cells in all analysed samples. The relationship between FOXP3 expression and clinicopathological features was assessed., Results: Among the studied patients, 14 (30.4%) were females and 32 (69.6%) were males, and the median age at diagnosis was 64.1 years. The median expression of FOXP3 was 84.9 positive cells/HPF. FOXP3 expression negatively correlated with Epstein-Barr virus-encoded small RNA positivity in tumour (p=0.041). The patients with low FOXP3 expression presented with aggressive clinical behaviour, including advance-staged diseases (p=0.043), splenomegaly (p=0.008), B symptoms (p=0.019) and extranodal involvement (p=0.019). The neutrophil-to-lymphocyte ratio was higher in the patients with low FOXP3 expression, compared with those with high FOXP3 expression. Low FOXP3 expression had an adverse effect on progression-free survival (PFS, p=0.033), and increased the risk of recurrence 2.320-fold (HR 2.320 (95% CI 1.109 to 4.856); p=0.025)., Conclusions: Patients with AITL with low FOXP3 expression tend to have aggressive clinical presentation and shortened PFS. These findings may help with risk stratification and determination of new treatment strategy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Alveolar capillary dysplasia with misalignment of the pulmonary veins (ACD/MPV) is a rare and lethal interstitial lung disorder, caused by a congenital abnormality affecting the development of the parenchyma and pulmonary vessels. We report the case of a newborn at the end of 40 weeks of pregnancy, who showed no cardiopulmonary anomalies in prenatal control ultrasounds. However, after delivery, pulmonary hypertension and hypoxemic respiratory failure became apparent. She died after 12 days from refractory hemodynamic and respiratory failure despite intensive therapy. A surgical lung biopsy and clinical autopsy were performed, both revealing the same histopathological signs consistent with this disorder. In our case, the findings of digestive and genital malformations, together with the genetic result of the alteration in the FOXF1 gene, led us to conclude the definitive diagnosis of alveolar capillary dysplasia., (Copyright © 2024 Sociedad Española de Anatomía Patológica. Publicado por Elsevier España, S.L.U. All rights reserved.)

Discoidin Domain Receptor 2 (DDR2) is a receptor tyrosine kinase for collagen, stimulating epithelial-mesenchymal transition and stiffness in breast cancer. Here, we investigated levels of DDR2 in breast tumor cells in relation to vascular invasion, TIL subsets, macrophages, molecular tumor subtypes, modes of detection and prognosis. This retrospective, population-based series of invasive breast carcinomas from the Norwegian Screening Program in Vestfold County (Norway), period 2004-2009, included 200 screening patients and 82 cases detected in screening intervals. DDR2 was examined on core needle biopsies using a semi-quantitative, immunohistochemical staining index and dichotomized as low or high DDR2 expression. Counts of macrophages and TIL subsets were dichotomized based on immunohistochemistry using TMA. We also recorded blood or lymphatic vessel invasion (BVI or LVI) as present or absent by immunohistochemistry. High expression of DDR2 in tumor cells showed significant relation with high counts of CD163+ macrophages (p < 0.001) and FOXP3 TILs (p = 0.011), presence of BVI (p = 0.028), high tumor cell proliferation by Ki67 (p = 0.033), ER negativity (p = 0.001), triple-negative cases (p = 0.038), basal-like features (p < 0.001) as well as interval detection (p < 0.001). By multivariate analysis, high DDR2 expression was related to reduced recurrence-free survival (HR, 2.3, p = 0.017), when examined together with histologic grading, lymph node assessment, tumor diameter, BVI, and molecular tumor subtype. This study supports a link between high DDR2 expression, high counts of macrophages by CD163 (tumor associated) and regulatory T cells by FOXP3 together with the presence of BVI, possibly indicating increased tumor motility and intravasation in aggressive breast tumors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Endometriosis's pathophysiology remains incompletely understood, with evidence pointing towards a dysregulated immune response. Regulatory T (Treg) cells, pivotal in maintaining self-tolerance, may facilitate the survival of ectopic endometrial cells within the abdominal cavity, thereby contributing to endometriosis development. This study aimed to assess the prevalence of CD39+CD73+ suppressor Treg cell subsets in the peripheral blood of endometriosis patients. This research focuses on the pivotal role of regulatory T-cells (Tregs), which are essential for maintaining immune tolerance and preventing autoimmune diseases. A case-control study was conducted, including 32 women diagnosed with endometriosis and 22 control subjects. The frequency of peripheral blood CD39+CD73+ suppressor Treg cells was quantified using flow cytometry. No significant differences were observed in the frequency of CD3+CD4+CD25High cells (Median [M]: 10.1; Interquartile Range [IQR]: 6.32‒18.3 vs. M: 9.72; IQR: 6.22-19.8) or CD3+CD4+CD25HighCD39+Foxp3+ cells (M: 31.1; IQR: 19.7-44.0 vs. M: 30.55; IQR: 18.5-45.5) between controls and patients. However, a significantly lower frequency of CD3+CD4+CD25HighCD39+CD73+ cells was observed in the endometriosis group compared to controls (M: 1.98; IQR: 0.0377-3.17 vs. M: 2.25; IQR: 0.50-4.08; p = 0.0483), suggesting a reduction in systemic immune tolerance among these patients. This finding highlights the potential role of CD39 and CD73 expression on Treg cells as biomarkers for assessing disease severity and progression. Furthermore, elucidating the mechanisms driving these alterations may unveil new therapeutic strategies to restore immune equilibrium and mitigate endometriosis symptoms., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier España, S.L.U.)

Tumor-infiltrating lymphocyte (TIL) density plays an important role in anti-tumor immunity and is associated with patient outcome in various human and canine malignancies. As a first assessment of the immune landscape of the tumor microenvironment in canine renal cell carcinoma (RCC), we retrospectively analyzed clinical data and quantified CD3, FoxP3, and granzyme B immunostaining in formalin-fixed paraffin-embedded tumor samples from 16 dogs diagnosed with renal cell carcinoma treated with ureteronephrectomy. Cell density was low for all markers evaluated. Increased numbers of intratumoral FoxP3 labelled (+) cells, as well as decreased granzyme B+: FoxP3+ TIL ratio, were associated with poor patient outcomes. Our initial study of canine RCC reveals that these tumors are immunologically cold and Tregs may play an important role in immune evasion., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Matthew Atherton reports financial support was provided by National Institutes of Health, NCI, K08CA252619. Charles-Antoine Assenmacher reports financial support was provided by National Institutes of Health Shared Instrumentation Grant (S10 OD023465–01A1). Charles-Antoine Assenmacher reports financial support was provided by Abramson Cancer Center (P30 CA016520). Jennifer Lenz reports financial support was provided by Miso Harris Fund donated to Penn Vet Comprehensive Cancer Care service. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Colon cancer is a heterogeneous disease and consists of various molecular subtypes. Despite advances in high-throughput expression profiling, limitations remain in predicting clinical outcome and assigning specific treatment to individual cases. Tumor-immune interactions play a critical role, with tumors that activate the immune system having better outcome for the patient. The localization of T cells within tumor epithelium, to enable direct contact, is essential for antitumor function, but bulk DNA/RNA sequencing data lacks spatial distribution information. In this study, we provide spatial T cell tumor distribution and connect these data with previously determined genomic data in the AC-ICAM colon cancer patient cohort., Methods: Colon cancer patients (n=90) with transcriptome data available were selected. We used a custom multiplex immunofluorescence assay on colon tumor tissue sections for quantifying T cell subsets spatial distribution in the tumor microenvironment, in terms of cell number, location, mutual distance, and distance to tumor cells. Statistical analyses included the previously determined Immunologic Constant of Rejection (ICR) transcriptome correlation and patient survival, revealing potential prognostic value in T cell spatial distribution., Results: T cell phenotypes were characterized and CD3 + CD8 - FoxP3 - T cells were found to be the predominant tumor-infiltrating subtype while CD3 + FoxP3 + T cells and CD3 + CD8 + T cells showed similar densities. Spatial distribution analysis elucidated that proliferative T cells, characterized by Ki67 expression, and Granzyme B-expressing T cells were predominantly located within the tumor epithelium. We demonstrated an increase in immune cell density and a decrease in the distance of CD3 + CD8 + T cells to the nearest tumor cell, in the immune active, ICR High, immune subtypes. Higher densities of stromal CD3 + FoxP3 + T cells showed enhanced survival outcomes, and patients exhibited superior clinical benefits when greater spatial distances were observed between CD3 + CD8 - FoxP3 - or CD3 + CD8 + T cells and CD3 + FoxP3 + T cells., Conclusion: Our study's in-depth analysis of the spatial distribution and densities of major T cell subtypes within the tumor microenvironment has provided valuable information that paves the way for further research into the intricate relationships between immune cells and colon cancer development., Competing Interests: DB reports employment with Kite, a Gilead Company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhu, Roelands, Ahmed, Stouten, Hoorntje, van Vlierberghe, Ijsselsteijn, Lei, de Miranda, Tollenaar, Vahrmeijer, Bedognetti, Hendrickx and Kuppen.)

Background: Among cutaneous squamous cell carcinomas, the ear (ecSCC) is one of the most common sites. Loco regional lymph node metastasis is found in six to eleven percent of cases, corresponding to increased metastasis compared to other sites. The aim of this study was to test the markers PD-L1, PD-1, CD4, CD8, and FoxP3 for suitability as prognostic predictive markers., Methods: Sixty-four patients with ecSCC were included in this study. The expression of immunohistochemical markers (PD-L1, PD-1, CD4, CD8, FOXP3) was correlated with retrospective clinic pathological parameters (lymph node metastasis, distant metastasis, lymph node metastasis during follow-up, disease progression, disease-specific death)., Results: There was a correlation between increased disease specific death and a weak Foxp3 (p = 0.003) or reduced CD8 (p = 0.04). A PD-L1 expression > 1% was found in 39.1% of patients., Conclusion: The investigated markers (CD4, CD8, FoxP3, PD-1, PD-L1) seem overall rather inappropriate for prognostic evaluation in ecSCC. Only the correlation of disease specific death with CD8 or FoxP3 seems to be worth testing in larger collectives., (© 2023. The Author(s).)

Regulatory T cells (Tregs) are a heterogeneous cell population that can either suppress or stimulate immune responses. Tumor-infiltrating Tregs are associated with an adverse outcome from most cancer types, but have generally been found to be associated with a good prognosis in colorectal cancer (CRC). We investigated the prognostic heterogeneity of Tregs in CRC by co-expression patterns and spatial analyses with diverse T cell markers, using multiplex fluorescence immunohistochemistry and digital image analysis in two consecutive series of primary CRCs (total n = 1720). Treg infiltration in tumors, scored as FOXP3 + or CD4 + /CD25 + /FOXP3 + (triple-positive) cells, was strongly correlated to the overall amount of CD3 + and CD8 + T cells, and consequently associated with a favorable 5-year relapse-free survival rate among patients with stage I-III CRC who underwent complete tumor resection. However, high relative expression of the activation marker CD25 in triple-positive Tregs was independently associated with an adverse outcome in a multivariable model incorporating clinicopathological and known molecular prognostic markers (hazard ratio = 1.35, p = 0.028). Furthermore, spatial marker analysis based on Voronoi diagrams and permutation testing of cellular neighborhoods revealed a statistically significant proximity between Tregs and CD8 + -cells in 18% of patients, and this was independently associated with a poor survival (multivariable hazard ratio = 1.36, p = 0.017). These results show prognostic heterogeneity of different Treg populations in primary CRC, and highlight the importance of multi-marker and spatial analyses for accurate immunophenotyping of tumors in relation to patient outcome., (© 2022. The Author(s).)

Background: Although the prognosis of patients experiencing recurrences after surgery for pancreatic cancer is extremely poor, patients who develop recurrence in the lung have a better prognosis compared to other types of recurrence. We performed a histo-immunological analysis of the metastatic specimens to identify specific features of this patient subgroup., Methods: We performed immunohistochemistry for CD4+, CD8+, CD45RO+, Foxp3, and PD-L1 in the lung (n = 22), peritoneal (n = 18), and liver (n = 6) metastases of pancreatic cancer. As microenvironmental and immunonutritional investigations, the tumor-stroma ratio and prognostic nutritional index (PNI) were utilized in the integrative analysis of immunological features., Results: We identified significantly increased tumor-infiltrating CD4+, CD8+, and CD45RO+ cells in lung metastasis, compared with peritoneal and liver metastases (lung vs. peritoneum/liver, CD4: P < 0.001/P = 0.015, CD8: P < 0.001/P = 0.038, CD45RO: P = 0.022/P = 0.012). The CD8/Foxp3 ratio was higher in the lung than in the liver (P = 0.024). PD-L1 expression was significantly higher in lung metastasis than in peritoneal metastasis (P = 0.010). Furthermore, we found that lung metastasis had fewer cancer stroma than peritoneal metastasis (P < 0.001). A higher PNI was observed in patients with lung metastasis, and PNI was positively correlated with tumor-infiltrating lymphocytes in metastatic sites., Conclusion: We identified that lung metastasis revealed an immunologically "hot" tumor with increased TILs and PD-L1 expression. This specific feature suggests that patients with lung metastasis can be candidates for immunotherapy, such as immune checkpoint inhibitors; therefore, our study provides a framework for developing individualized treatment strategies for this patient subgroup., (© 2022. The Author(s) under exclusive licence to Japan Society of Clinical Oncology.)

Purpose: To date, many genes have been associated with congenital hypothyroidism (CH). Our aim was to identify the mutational spectrum of 23 causative genes in Turkish patients with permanent CH, including thyroid dysgenesis (TD) and dyshormonogenesis (TDH) cases., Methods: A total of 134 patients with permanent CH (130 primary, 4 central) were included. To identify the genetic etiology, we screened 23 candidate genes associated with CH by next-generation sequencing. For confirmation and to detect the status of the specific familial variant in relatives, Sanger sequencing was also performed., Results: Possible pathogenic variants were found in 5.2% of patients with TD and in 64.0% of the patients with normal-sized thyroid or goiter. In all patients, variants were most frequently found in TSHR, followed by TPO and TG. The same homozygous TSHB variant (c.162 + 5G > A) was identified in four patients with central CH. In addition, we detected novel variants in the TSHR, TG, SLC26A7, FOXE1, and DUOX2., Conclusion: Genetic causes were determined in the majority of CH patients with TDH, however, despite advances in genetics, we were unable to identify the genetic etiology of most CH patients with TD, suggesting the effect of unknown genes or environmental factors. The previous studies and our findings suggest that TSHR and TPO mutations is the main genetic defect of CH in the Turkish population., (© 2021. Italian Society of Endocrinology (SIE).)

Programmed death 1 (PD1)/its ligand PD-L1 concomitant with T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3)/its ligand galectin 9 (Gal-9) and the forkhead box P3 (FOXP3) might be involved in tolerance after liver transplantation (LT). Liver biopsies from 38 tolerant, 19 nontolerant (including 16 samples that triggered reintroduction of immunosuppression [IS] and 19 samples after IS reintroduction), and 38 control LT patients were studied. The expressions of PD1, PD-L1, Gal-9, and FOXP3 were determined by immunohistochemical and immunofluorescence (IF) staining. The success period of IS withdrawal was calculated using Kaplan-Meier curve analysis. Tolerant and control patients exhibited higher PD-L1, Gal-9, and FOXP3 levels than nontolerant patients at the moment of triggering IS reintroduction. High expressions of PD-L1 and Gal-9 were associated with prolonged success of tolerance (83.3% versus 36.7% [P < 0.01] and 73.1% versus 42.9% [P = 0.03]). A strong correlation between PD-L1 and Gal-9 expression levels was detected (Spearman r = 0.73; P ≤ 0.001), and IF demonstrated colocalization of PD-L1 and Gal-9 in the cytoplasm of hepatocytes. In conclusion, the present study demonstrated that increased expressions of PD-L1 and Gal-9 were associated with sustained tolerance after IS withdrawal in pediatric liver transplantation., (Copyright © 2021 American Association for the Study of Liver Diseases.)

Background: The prognostic value of intratumor T regulatory cells (Tregs) in colorectal cancer (CRC) was previously reported, but the role of these cells in tumor draining lymph nodes (TDLNs) was less addressed., Methods: A total of 150 CRC stages I-IV were retrospectively enrolled. Intratumor and TDLN Tregs were examined by immunohistochemical assay. The association of these cells was estimated by Pearson correlation. Survival analyses of subgroups were conducted by Kaplan-Meier curves, and the log-rank test and risk factors for survival were tested by the Cox proportional hazard model., Results: High accumulation of Tregs in tumors was significant in patients with younger age and good histological grade, where enrichment of these cells in TDLNs was more apparent in those with node-negative disease and early TNM stage disease, both of which were more common in early T stage cases. A significant correlation of intratumoral and TDLN Tregs was detected. Patients with higher intratumoral Tregs displayed significantly better PFS and OS than those with lower Tregs. However, no such differences were found, but a similar prognostic prediction trend was found for these cells in TDLNs. Finally, intratumoral Tregs were an independent prognostic factor for both PFS (HR = 0.97, 95% CI 0.95-0.99, P < 0.01) and OS (HR = 0.98, 95% CI 0.95-1.00, P = 0.04) in the patients., Conclusions: Higher intratumor Tregs were associated with better survival in CRC. Although no such role was found for these cells in TDLNs, the positive correlation and similar prognostic prediction trend with their intratumoral counterparts may indicate a parallelized function of these cells in CRC., (© 2022. The Author(s).)

Introduction: Tumor-infiltrating lymphocytes represent a pivotal component of the host anti-tumor response. Thus, they considerably influence the evolution of cancers including non-small cell lung carcinomas. Even if, this important role is consensual, many discordant results are published in the literature about the prognostic role of the different populations of tumor-infiltrating lymphocytes. The aim of our work was to evaluate the prognostic impact of CD8+, CD4+, and forkhead box protein P3+ lymphocytes in the tumor microenvironment of non-small cell lung carcinomas., Methods: We conducted a retrospective descriptive study, which included non-small cell lung carcinomas diagnosed in the department of pathology and followed in the medical oncology department of the same hospital between 2011 and 2015. Tumor-infiltrating lymphocytes were analyzed by the immunohistochemical method for forkhead box protein P3, CD4, and CD8. Intratumoral and stromal-labeled lymphocytes were quantified by manual counting at high magnification (×400). Forkhead box protein P3+/CD8+, forkhead box protein P3+/CD4+, and CD8+/CD4+ ratios were subsequently calculated. The prognostic value of tumor-infiltrating lymphocytes was assessed in respect of overall survival, recurrence-free survival, and relapse-free survival., Results: Thirty-nine patients were included. The mean age of patients was 59.6 years. A complete surgical resection ( p  = 0.009), and a CD8/CD4 ratio ( p  = 0.008) were prognostic factors for overall survival. Complete surgical resection ( p  = 0.003), the forkhead box protein P3/CD8 ( p  = 0.005), and forkhead box protein P3/CD4 ( p  = 0.037) ratios were prognostic factors for recurrence-free survival. The CD8+ tumor-infiltrating lymphocytes rate ( p  = 0.037) was a prognostic factor for relapse-free survival with a threshold of 67.8/high power field. Microscopic subtype ( p  = 0.037) was a prognostic factor for relapse-free survival when only adenocarcinoma and squamous cell carcinoma were considered. In multivariate analysis, age ( p  = 0.004) and a CD8/CD4 ratio ( p  = 0.016) were independent predictors of overall survival., Conclusion: Despite the limitations of our study, our results confirm the prognostic value of tumor-infiltrating lymphocytes in non-small cell lung carcinomas and the importance of the combined quantification of their different subpopulations.

Treg therapies are being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs remains controversial. We challenged human Tregs ex-vivo with pro-inflammatory cytokines IL-6 and TNF α and observed greatly enhanced proliferation stimulated by anti-CD3 and anti-CD28 (aCD3/28) beads or CD28 superagonist (CD28SA). The cytokine-exposed Tregs maintained high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low IFNγ, IL-4, and IL-17 secretion. Blocking TNF receptor using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression. These results prompted us to consider using CD28SA together with IL-6 and TNF α without aCD3/28 beads (beadless) as an alternative protocol for therapeutic Treg manufacturing. Metabolomics profiling revealed more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential during beadless Treg expansion. Finally, beadless expanded Tregs maintained suppressive functions in vitro and in vivo . These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function. This property can be harnessed for therapeutic Treg manufacturing., Competing Interests: QT is a co-founder and scientific advisor of Sonoma Biotherapeutics. QT, NS, and FV are co-inventors of a patent on manufacturing Tregs based on results from this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Skartsis, Peng, Ferreira, Nguyen, Ronin, Muller, Vincenti and Tang.)

Background: Regulatory T cells (Tregs) are important in regulating responses to innocuous antigens, such as allergens, by controlling the Th2 response, a mechanism that appears to be compromised in atopic asthmatic individuals. Different isogenic mouse strains also have distinct immunological responses and susceptibility to the experimental protocols used to develop lung allergic inflammation. In this work, we investigated the differences in the frequency of Treg cell subtypes among A/J, BALB/c, and C57BL/6, under normal conditions and following induction of allergic asthma with ovalbumin (OVA)., Methods: Subcutaneous sensitization followed by 4 consecutive intranasal OVA challenges induced asthma characteristic changes such as airway hyperreactivity, inflammation, and production of Th2 cytokines (IL-4, IL-13, IL-5, and IL-33) in the lungs of only A/J and BALB/c but not C57BL/6 strain and evaluated by invasive whole-body plethysmography, flow cytometry, and ELISA, respectively., Results: A/J strain naturally showed a higher frequency of CD4 + IL-10 + T cells in the lungs of naïve mice compared to the other strains, accompanied by higher frequencies of CD4 + IL-4 + T cells. C57BL/6 mice did not develop lung inflammation and presented higher frequency of CD4 + CD25 + Foxp3 + Treg cells in the bronchoalveolar lavage fluid (BALF) after the allergen challenge. In in vitro settings, allergen-specific stimulation of mediastinal LN (mLN) cells from OVA-challenged animals induced higher frequency of CD4 + IL-10 + Treg cells from A/J strain and CD4 + CD25 + Foxp3 + from C57BL/6., Conclusions: The observed differences in the frequencies of Treg cell subtypes associated with the susceptibility of the animals to experimental asthma suggest that CD4 + CD25 + Foxp3 + and IL-10-producing CD4 + Treg cells may play different roles in asthma control. Similar to asthmatic individuals, the lack of an efficient regulatory response and susceptibility to the development of experimental asthma in A/J mice further suggests that this strain could be preferably chosen in experimental models of allergic asthma., Competing Interests: There are no conflicts of interest for all named author., (Copyright © 2021 C. T. Azevedo et al.)

Luminal A breast cancers are generally associated with low metastatic potential and good prognosis. However, there is a proportion of patients, who present with metastases in lymph nodes. The aim of our study was to determine the association between the number of positive lymph nodes and infiltrates of tumor-associated cytotoxic CD8 + (CTLs), regulatory FOXP3 + T cells (Tregs), as well as other prognostic factors. Immunohistochemistry (IHC) for CD8 + and FOXP3 + was performed in 87 formalin-fixed paraffin-embedded primary breast cancer tissues, and cell infiltrate was assessed under light microscope. We observed that node-positive cases were associated with higher numbers of Treg cells and lower CTL/Treg ratio. There was also an inverse correlation between the CTL/Treg ratio and the number of metastatic lymph nodes. Similar relationships were found between the number of metastatic lymph nodes and Treg density or CTL/Treg ratio in pT1 BC. An elevated intratumoral CTL/Treg ratio was associated with pN0 stage. The relationship between lymphovascular invasion (LVI) and Treg density was also noted in node-negative tumors. In addition, more advanced nodal stage was related to LVI, higher pT, and lower PR expression. The numbers of CD8 + and FOXP3 + were also associated with tumor size, histologic grade, PR expression, and mitotic index. The results of our study suggested that the levels of tumor-infiltrating regulatory and cytotoxic cells as well as the balance between them play a role in lymphovascular spread of luminal A breast cancers., (© 2021. The Author(s).)

UVR and immunosuppression are major risk factors for cutaneous squamous cell carcinoma (cSCC). Regulatory T cells promote cSCC carcinogenesis, and in other solid tumors, infiltrating regulatory T cells and CD8 + T cells express ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) (also known as CD39), an ectoenzyme that catalyzes the rate-limiting step in converting extracellular adenosine triphosphate (ATP) to extracellular adenosine (ADO). We previously showed that extracellular purine nucleotides influence DNA damage repair. In this study, we investigate whether DNA damage repair is modulated through purinergic signaling in cSCC. We found increased ENTPD1 expression on T cells within cSCCs when compared with the expression on T cells from blood or nonlesional skin, and accordingly, concentrations of derivative extracellular adenosine diphosphate (ADP), adenosine monophosphate (AMP), and ADO are increased in tumors compared with those in normal skin. Importantly, ENTPD1 expression is significantly higher in human cSCCs that metastasize than in those that are nonmetastatic. We also identify in a mouse model that ENTPD1 expression is induced by UVR in an IL-27-dependent manner. Finally, increased extracellular ADO is shown to downregulate the expression of NAP1L2, a nucleosome assembly protein we show to be important for DNA damage repair secondary to UVR. Together, these data suggest a role for ENTPD1 expression on skin-resident T cells to regulate DNA damage repair through purinergic signaling to promote skin carcinogenesis and metastasis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Immunoprofiling has an established impact on the prognosis of several cancers; however, its role and definition in high-grade serous ovarian cancer (HGSOC) are mostly unknown. This study is to investigate immunoprofiling which could be a prognostic factor in HGSOC. We produced tumor microarrays of 187 patients diagnosed with HGSOC. We performed a multiplexed immunofluorescence staining using Opal Multiplex IHC kit and quantitative analysis with Vectra-Inform system. The expression intensities of programmed death-ligand 1 (PD-L1), CD4, CD8, CD20, FoxP3, and CK in whole tumor tissues were evaluated. The enrolled patients showed general characteristics, mostly FIGO stage III/IV and responsive to chemotherapy. Each immune marker showed diverse positive densities, and each tumor sample represented its immune characteristics as an inflamed tumor or noninflamed tumor. No marker was associated with survival as a single one. Interestingly, high ratios of CD8 to FoxP3 and CD8 to PD-L1 were related to the favorable overall survival (77 vs. 39 months, 84 vs. 47 months, respectively), and CD8 to PD-L1 ratio was also a significant prognostic factor (HR 0.621, 95% CI 0.420-0.917, p = 0.017) along with well-known clinical prognostic factors. Additionally, CD8 to PD-L1 ratio was found to be higher in the chemosensitive group ( p = 0.034). In conclusion, the relative expression levels of CD8, FoxP3, and PD-L1 were significantly related to the clinical outcome of patients with HGSOC, which could be a kind of significant immunoprofiling of ovarian cancer patients to apply for treatment.

Postmeiotic germ cells require the lactate produced by the adjacent Sertoli cells (SCs) as their sole energy fuels. Lactate production in SCs is elaborately regulated by monitoring the transcription of the lactate dehydrogenase A (Ldha) gene. However, the transcription factors that are responsible for the control of Ldha transcription in SCs remain ill defined. Herein, the expression of forkhead box Q1 (FOXQ1), a central modulator of glucose metabolism in liver, was demonstrated in mouse testis throughout postnatal development, with maximum levels in adult specimens. At this age, FOXQ1 was immunolocalized in the nuclei of the functionally mature SCs. Testicular levels of FOXQ1 were overtly modulated by germ cells (GCs)-derived IL-1α, in a dose- and time-dependent manner. To further clarify the biological functions of FOXQ1, we disrupted the mouse Foxq1 gene using a Cas9/RNA-mediated gene targeting strategy. Foxq1 -/- males were subfertile and showed oligoasthenozoospermia due to lactate deficiency. Moreover, we provided the molecular evidence that FOXQ1 may regulate lactate production by directly targeting the transactivation of the Ldha gene in SCs. From a functional standpoint, overexpression of the exogenous Ldha ameliorated Foxq1 deficiency-impaired lactate synthesis in the SCs Foxq1-/- cells. Thus, these findings collectively underscore a reproductive facet of this recently characterized transcription factor, which may operate as a novel transcriptional integrator linking energy homeostasis and nursery function in SCs., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Regulatory T cells (Tregs) play a key role in the progression of tumors. These cells express forkhead box P3 (FOXP3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4), which are the potential targets for cancer immunotherapy. The present study aimed to evaluate FOXP3 and CTLA4 transcripts in patients with bladder cancer (BC) compared with healthy individuals., Methods: Transcripts of CTLA4 and FOXP3 genes in the peripheral blood mononuclear cells (PBMCs) of 50 patients with histologically confirmed BC and 50 healthy individuals were assessed at the Institute for Cancer Research, Shiraz University of Medical Sciences (Shiraz, Iran) during 2014-2016. RNA was extracted from PBMCs, then cDNA was synthesized and subjected to quantitative real-time PCR (qRT-PCR) using appropriate primers. Statistical analysis was performed using SPSS software (version 21.0)., Results: Significantly higher amounts of CTLA4 and FOXP3 gene transcripts were found in the peripheral blood of BC patients compared with healthy individuals. The expression of both genes was significantly higher in patients with non-invasive and grade I/II BC. The median of CTLA4 and FOXP3 transcript expressions was 3.74 and 5.39, respectively, in non-invasive BC patients, which was significant compared with the control group (P=0.0016 and P=0.009, respectively). The median of target gene mRNA expression in grade I/II BC patients was 2.9 for CTLA4 and 6.61 for FOXP3 , which was significant compared with the controls (P=0.013 and P=0.0037, respectively)., Conclusion: This study highlights the functional activity of Tregs in early stages of bladder cancer and showed the importance of CTLA4 and FOXP3, when it comes to screening BC., Competing Interests: Conflict of Interest: None declared., (Copyright: © Iranian Journal of Medical Sciences.)

Intrahepatic cholangiocarcinoma (ICC) is highly invasive and carries high mortality due to limited therapeutic strategies. In other solid tumors, immune checkpoint inhibitors (ICIs) target cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD1), and the PD1 ligand PD-L1 has revolutionized treatment and improved outcomes. However, the relationship and clinical significance of CTLA-4 and PD-L1 expression in ICC remains to be addressed. Deciphering CTLA-4 and PD-L1 interactions in ICC enable targeted therapy for this disease. In this study, immunohistochemistry (IHC) was used to detect and quantify CTLA-4, forkhead box protein P3 (FOXP3), and PD-L1 in samples from 290 patients with ICC. The prognostic capabilities of CTLA-4, FOXP3, and PD-L1 expression in ICC were investigated with the Kaplan-Meier method. Independent risk factors related to ICC survival and recurrence were assessed by the Cox proportional hazards models. Here, we identified that CTLA-4 + lymphocyte density was elevated in ICC tumors compared with peritumoral hepatic tissues ( P <.001), and patients with a high density of CTLA-4 + tumor-infiltrating lymphocytes (TILs CTLA-4 High ) showed a reduced overall survival (OS) rate and increased cumulative recurrence rate compared with patients with TILs CTLA-4 Low ( P <.001 and P = .024, respectively). Similarly, patients with high FOXP3 + TILs (TILs FOXP3 High ) had poorer prognoses than patients with low FOXP3 + TILs ( P  = .021, P = .034, respectively), and the density of CTLA-4 + TILs was positively correlated with FOXP3 + TILs (Pearson r = .31, P <.001). Furthermore, patients with high PD-L1 expression in tumors (Tumor PD-L1 High ) and/or TILs CTLA-4 High presented worse OS and a higher recurrence rate than patients with TILs CTLA-4 Low Tumor PD-L1 Low . Moreover, multiple tumors, lymph node metastasis, and high Tumor PD-L1 /TILs CTLA-4 were independent risk factors of cumulative recurrence and OS for patients after ICC tumor resection. Furthermore, among ICC patients, those with hepatolithiasis had a higher expression of CTLA-4 and worse OS compared with patients with HBV infection or undefined risk factors (P = .018). In conclusion, CTLA-4 is increased in TILs in ICC and has an expression profile distinct from PD1/PD-L1. Tumor PD-L1 /TILs CTLA-4 is a predictive factor of OS and ICC recurrence, suggesting that combined therapy targeting PD1/PD-L1 and CTLA-4 may be useful in treating patients with ICC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Guo, Lu, Zeng, Zhou, Sun, Yang, Pei, Meng, Shen, Zhang, Cai, Huang, Ke, Shi, Zhou, Fan, Chen, Yang, Shi and Huang.)

Introduction: CD4 + T cells with regulatory function co-expressing Foxp3 and RORγt are linked to the development of oral tolerance towards innocuous food antigens in mice. This study aimed to discern the role played by IL-6 and retinoic acid (RA) in the in vitro generation of Foxp3 + RORγt + T cells and to investigate whether such cells have suppressive properties., Methods: CD4 + CD25 - T cells isolated from the spleen of BALB/c mice, were stimulated in the presence of IL-2 alone or together with TFG-β and different concentrations of IL-6 and/or RA. Percentage of Foxp3 + , RORγt + , IL-17 + , Foxp3 + RORγt - , Foxp3 + RORγt + , and Foxp3 - RORγt + T cells within the total CD4 + T cell population, production of cytokines (IL-10 and IL-17A) and gene expression ( Foxp3, Rorc, Tgfb1, Il6, Il10 , and Il17 ) were assessed at different time points. The phenotype and ability of cells generated from CD4 + CD44 - CD62L + cells in the presence of RA to suppress effector T cell proliferation was assessed., Results: TGF-β plus IL-6 induced the generation of Foxp3 + and double positive Foxp3 + RORγt + T cells to a higher extent than TGF-β alone at the beginning of the incubation period, although expression of Foxp3 subsequently declined. RA, added to TGF-β, increased Foxp3 and Rorc expression and Foxp3 and RORγt transcription and promoted the differentiation of Foxp3 + RORγt - and Foxp3 + RORγt + cells that expressed and secreted IL-17. Foxp3 + T cells generated in vitro in presence of RA were functionally suppressive., Conclusions: Under the influence of IL-2 and TGF-β, suppressive Foxp3 + RORγt + T cells that express and secrete IL-17 can be produced in vitro and RA further contributes to stabilize this phenotype., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Martínez-Blanco, Lozano-Ojalvo, Pérez-Rodríguez, Benedé, Molina and López-Fandiño.)

The retinoid X receptor agonist bexarotene promotes remyelination in patients with multiple sclerosis. Murine studies have also demonstrated that RXR agonists have anti-inflammatory effects by enhancing the ability of all-trans-retinoic acid ( at RA) to promote T-regulatory cell (Treg) induction and reduce Th17 differentiation in vitro. By stimulating human naïve CD4 T-cells in the presence of Treg or Th17 skewing cytokines, we show that bexarotene also tips the human Treg/Th17 axis in favor of Treg induction, but unlike murine cells this occurs independently of at RA and retinoic acid receptor signaling. Tregs induced in the presence of bexarotene express canonical markers of T-regulation and are functionally suppressive in vitro. Circulating Treg numbers did not increase in the blood of trial patients receiving bexarotene; we believe this is because Treg induction is likely to occur within tissues. These findings lend support to developing RXR agonists as treatments of autoimmune diseases, in particular multiple sclerosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gaunt, Rainbow, Mackenzie, Jarvis, Mousa, Cunniffe, Georgieva, Brown, Coles and Jones.)

Immunosuppressive drugs such as cyclosporine A (CSA) can disrupt thymic structure and functions, ultimately inducing syngeneic/autologous graft-versus-host disease together with involuted medullas. To elucidate the effects of CSA on the thymus more precisely, we analyzed the effects of CSA on the thymus and T cell system using rats. In addition to confirming the phenomena already reported, we newly found that the proportion of recent thymic emigrants also greatly decreased, suggesting impaired supply. Immunohistologically, the medullary thymic epithelial cells (mTECs) presented with a relative decrease in the subset with a competent phenotype and downregulation of class II major histocompatibility complex molecules. In control rats, thymic dendritic cells (DCs) comprised two subsets, XCR1 + SIRP1α - CD4 - and XCR1 - SIRP1α + CD4 + . The former had a tendency to selectively localize in the previously-reported epithelium-containing areas of the rat medullas, and the number was significantly reduced by CSA treatment. The epithelium-free areas, another unique domains in the rat medullas, contained significantly more Foxp3 + thymic Tregs. With CSA treatment, the epithelium-free areas presented strong involution, and the number and distribution of Tregs in the medulla were greatly reduced. These results suggest that CSA inhibits the production of single-positive thymocytes, including Tregs, and disturbs the microenvironment of the thymic medulla, with a decrease of the competent mTECs and disorganization of epithelium-free areas and DC subsets, leading to a generation of autoreactive T cells with selective medullary involution., (© 2021. The Author(s).)

Background: Alveolar capillary dysplasia with or without misalignment of the pulmonary veins (ACD/MPV) is a lethal congenital lung disorder associated with a variety of heterozygous genomic alterations in the FOXF1 gene or its 60 kb enhancer. Cases without a genomic alteration in the FOXF1 locus have been described as well. The mechanisms responsible for FOXF1 haploinsufficiency and the cause of ACD/MPV in patients without a genomic FOXF1 variant are poorly understood, complicating the search for potential therapeutic targets for ACD/MPV. To investigate the contribution of aberrant DNA methylation, genome wide methylation patterns of ACD/MPV lung tissues were compared with methylation patterns of control lung tissues using the recently developed technique Methylated DNA sequencing (MeD-seq)., Results: Eight ACD/MPV lung tissue samples and three control samples were sequenced and their mutual comparison resulted in identification of 319 differentially methylated regions (DMRs) genome wide, involving 115 protein coding genes. The potentially upregulated genes were significantly enriched in developmental signalling pathways, whereas potentially downregulated genes were mainly enriched in O-linked glycosylation. In patients with a large maternal deletion encompassing the 60 kb FOXF1 enhancer, DNA methylation patterns in this FOXF1 enhancer were not significantly different compared to controls. However, two hypermethylated regions were detected in the 60 kb FOXF1 enhancer of patients harbouring a FOXF1 point mutation. Lastly, a large hypermethylated region overlapping the first FOXF1 exon was found in one of the ACD/MPV patients without a known pathogenic FOXF1 variation., Conclusion: This is the first study providing genome wide methylation data on lung tissue of ACD/MPV patients. DNA methylation analyses in the FOXF1 locus excludes maternal imprinting of the 60 kb FOXF1 enhancer. Hypermethylation at the 60 kb FOXF1 enhancer might contribute to FOXF1 haploinsufficiency caused by heterozygous mutations in the FOXF1 coding region. Interestingly, DNA methylation analyses of patients without a genomic FOXF1 variant suggest that abnormal hypermethylation of exon 1 might play a role in some ACD/MPV in patients., (© 2021. The Author(s).)

Post-transplant lymphoproliferative disorder (PTLD) is a life-threatening complication of transplantation. In addition to reactivation of Epstein-Barr virus in immunocompromised patients, impaired tumor immunity is suggested to be a risk factor for PTLD. However, it remains unclear whether immune suppressive tumor-infiltrating lymphocytes (TILs) correlate with the occurrence or prognosis of PTLD. We analyzed TILs in 26 patients with PTLD to elucidate the clinicopathological significance of the expression of PD-1 and FoxP3, which are associated with exhausted T-cells and regulatory T-cells (Tregs), respectively. Numbers of PD-1 + TILs in the PTLD specimens were significantly higher in patients who developed PTLD early after transplantation (P = 0.0040), while numbers of FoxP3 + TILs were not (P = 0.184). There was no difference in overall response rate regardless of the expression of PD-1 or FoxP3. FoxP3 high patients tended to have a shorter time to progression compared with FoxP3 low patients, especially in the case of FoxP3 high patients with diffuse large B-cell lymphoma-subtype PTLD (P = 0.011), while PD-1 high patients did not. These results suggest that T-cell exhaustion may be mainly associated with PTLD development, while immune suppression by Tregs may be dominant in enhanced progression of PTLD following disease occurrence.

Next-generation tissue-based biomarkers for immunotherapy will likely include the simultaneous analysis of multiple cell types and their spatial interactions, as well as distinct expression patterns of immunoregulatory molecules. Here, we introduce a comprehensive platform for multispectral imaging and mapping of multiple parameters in tumor tissue sections with high-fidelity single-cell resolution. Image analysis and data handling components were drawn from the field of astronomy. Using this "AstroPath" whole-slide platform and only six markers, we identified key features in pretreatment melanoma specimens that predicted response to anti-programmed cell death-1 (PD-1)-based therapy, including CD163 + PD-L1 - myeloid cells and CD8 + FoxP3 + PD-1 low/mid T cells. These features were combined to stratify long-term survival after anti-PD-1 blockade. This signature was validated in an independent cohort of patients with melanoma from a different institution., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Gastric cancer (GC) is mostly diagnosed at advanced stage, so prognosis is poor. Therefore, it is necessary to understand the molecular mechanism of GC development to design new targeted treatment to improve the prognosis of gastric cancer patients., Aim of the Work: To assess the prognostic value of NEDD-9 and FOXL-1 expression in intestinal type gastric cancer patients, as well as their relationship to clinicopathologic features of the disease and patients outcome., Patients and Methods: This is a retrospective study; we included 50 sections from formalin-fixed, paraffin-embedded tissue samples which included intestinal type GC and adjacent non-neoplastic gastric mucosa in the same block that were subjected to immunohistochemistry with anti-NEDD-9 and anti-FOXL-1 antibody. Patients were retrospectively followed up for about 5 years for assessment of tumor progression and survival in relation to marker expression., Results: High NEDD-9 and low FOXL-1 expression were found in intestinal type GC more than adjacent non-neoplastic mucosa (p < 0.001). NEDD-9 high expression and FOXL-1 low expression were associated with presence of helicobacter pylori gastritis (p = 0.010, 0.049), high grade (p = 0.007, 0.004), high stage (p < 0.001), presence of distant metastases (p = 0.029, 0.021), poor DFS (p = 0.003), and OS rates (< 0.001)., Conclusion: NEDD-9 overexpression and FOXL-1 deficiency in intestinal type GC can help in prediction of tumor prognosis and it can guide the selection of patients for future therapies in gastric carcinoma.

The association of immune markers and clinicopathologic features and patient outcome has not been extensively studied in Merkel cell carcinoma (MCC). We correlated tumoral PD-L1 and IDO1 expression, and intratumoral CD8+ and FoxP3+ lymphocytes count with clinicopathologic variables, Merkel cell polyomavirus (MCPyV) status, and patient outcomes in a series of 132 MCC. By univariate analyses, tumoral PD-L1 expression >1% and combined tumoral PD-L1 >1% and high intratumoral FoxP3+ lymphocyte count correlated with improved overall survival (OS) ( p = 0.016, 0.0072), MCC-specific survival (MSS) ( p = 0.019, 0.017), and progression-free survival (PFS) ( p = 0.043, 0.004, respectively). High intratumoral CD8+ and FoxP3+ lymphocyte count correlated with longer MSS ( p = 0.036) and improved PFS ( p = 0.047), respectively. Ulceration correlated with worse OS and worse MSS. Age, male gender, and higher stage (3 and 4) significantly correlated with worse survival. MCPyV positivity correlated with immune response. By multivariate analyses, only ulceration and age remained as independent predictors of worse OS; gender and stage remained for shorter PFS. Tumoral PD-L1 expression and increased density of intratumoral CD8+ lymphocytes and FoxP+ lymphocytes may represent favorable prognosticators in a subset of MCCs. Tumoral PD-L1 expression correlated with intratumoral CD8+ and FoxP3+ lymphocytes, which is supportive of an adaptive immune response.

Forkhead box P3 (Foxp3) expressing CD4 + CD25 + regulatory T cells (Tregs), an essential subset of immune T cells for maintaining immune homeostasis is implicated as a negative regulator in an anti-tumor immune response. Current researches suggest that reducing tumor-infiltrating Tregs contribute to enhanced anti-cancer effect. However, the mechanism of infiltration of a large number of Tregs into tumor tissues is still unclear. In this study, human induced Tregs (iTregs) were co-cultured with human hepatocytes and various types of cancer cells (HepG2, NSCLC, and AsPC-1) supernatants. Foxp3, multiple cytokines, levels of apoptosis and suppressive ability of iTregs were detected by FACS. Western blot was employed to test of proteins. Impact of HepG2 supernatants on T cell subpopulations differentiation, cytokines in supernatants were examed by FACS and ELISA respectively. Anti-IL-10R antibody and JAK1 inhibitor were used to reconfirm the role of tumor-derived IL-10 play in the regulation on iTregs. Hepatocarcinoma cells (HCC) supernatants treatment increases Foxp3 stability and reduces apoptosis level in human iTregs without influencing its differentiation trend. Furthermore, IL-10 was found to be extremely higher in HCC supernatants than other groups, IL-10R blockade neutralize the effect of HCC supernatants on iTregs in vitro obviously. HCC supernatants also reversed IL-1β/6 triggered decline on Foxp3 which may be related to higher expression of JAK1 and elevated phosphorylation level of STAT5 induced by IL-10. Our results suggest that improved stability and abnormal accumulation of Tregs in tumor microenvironment is IL-10/JAK1/STAT5 signal pathway-dependent and provide a novel approach for improving the efficiency of anti-tumor immunotherapy., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Immunotherapies may prolong the survival of patients with small-cell lung cancer (SCLC) to some extent. The role of forkhead box protein P3 (FOXP3) in tumor microenvironment (TME) remains controversial. We aimed to examine FOXP3-related expression characteristics and prognostic values and to develop a clinically relevant predictive system for SCLC., Methods: We enrolled 102 patients with histologically confirmed SCLC at stages I-III. Through immunohistochemistry, we determined the expression pattern of FOXP3 and its association with other immune biomarkers. By machine learning and statistical analysis, we constructed effective immune risk score models. Furthermore, we examined FOXP3-related enrichment pathways and TME traits in distinct cohorts., Results: In SCLC, FOXP3 level was significantly associated with status of programmed death-ligand 1 (PD-L1), programmed cell death protein 1 (PD-1), CD4, CD8, and CD3 (p=0.002, p=0.001, p=0.002, p=0.030, and p<0.001). High FOXP3 expression showed longer relapse-free survival (RFS) than the low-level group (41.200 months, 95% CI 26.937 to 55.463, vs 14.000 months, 95% CI 8.133 to 19.867; p=0.008). For tumor-infiltrating lymphocytes (TILs), subgroup analysis demonstrated FOXP3 and PD-1, PD-L1, lymphocyte activation gene-3, CD3, CD4, or CD8 double positive were significantly correlated with longer RFS. We further performed importance evaluation for immune biomarkers, constructed an immune risk score incorporating the top three important biomarkers, FOXP3, TIL PD-L1, and CD8, and found their independently prognostic role to predict SCLC relapse. Better predictive performance was achieved in this immune risk model compared with single-indicator-based or two-indicator-based prediction systems (area under the curve 0.715 vs 0.312-0.711). Then, relapse prediction system integrating clinical staging and immune risk score was established, which performed well in different cohorts. High FOXP3-related genes were enriched in several immune-related pathways, and the close relationships of interleukin-2, CD28 , basic excision repair genes MUTYH , POLD1 , POLD2 , and oxidative phosphorylation related gene cytochrome c oxidase subunit 8A with FOXP3 expression were revealed. Moreover, we found low-immune risk score group had statistically higher activated CD4 + memory T cells (p=0.014) and plasma cells (p=0.049) than the high-risk group. The heterogeneity of tumor-infiltrating immune cells might represent a promising feature for risk prediction in SCLC., Conclusion: FOXP3 interacts closely with immune biomarkers on tumor-infiltrating cells in TME. This study highlighted the crucial prognostic value and promising clinical applications of FOXP3 in SCLC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Background: Helicobacter pylori infection is the main cause of chronic gastritis in children. Little is known about the effect of Helicobacter pylori on microbiota and immunity. This study was aimed at characterizing stomach microbiota and immune-regulatory properties of children with Helicobacter pylori colonization., Methods: We studied 122 children who had undergone gastric endoscopy due to gastrointestinal symptoms, 57 were diagnosed with Helicobacter pylori infection. Endoscopic mucosal biopsy samples were obtained for DNA and RNA extraction. Microbiomes were analyzed by 16S rRNA profiling, with the differentially expressed genes analyzed using RNA sequencing. The RNA-sequencing results of selected genes were validated by qRT-PCR., Results: Bacterial diversity of Helicobacter pylori -positive gastric specimens were lower than those of negative, and both groups were clearly separated according to beta diversity. Helicobacter pylori -positive group significantly reduced proportions of six phyla and eight genera; only Helicobacter taxa were more abundant in Helicobacter pylori- negative group. Gastric tissues RNA sequencing showed increased expression of multiple immune response genes in Helicobacter pylori -infection. Helicobacter pylori -infected children with restructured gastric microbiota had higher levels of FOXP3, IL-10, TGF-β1 and IL-17A expressions, which were consistent with increased CD4 + T cell and macrophagocyte, compared with non-infected children., Conclusions: Presence of Helicobacter pylori significantly influences gastric microbiota and results in lower abundance of multiple taxonomic levels in children. Meanwhile, it affects gastric immune environment and promotes the occurrence of gastritis., Clinical Trial Registration: [http://www.chictr.org.cn], identifier [ChiCTR1800015190]., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zheng, Miao, Luo, Long, Chen, Shu, Gu, Peng, Li, Zhao, Botchway, Fang and Jiang.)

The parabrachial nucleus (PB) is a complex structure located at the junction of the midbrain and hindbrain. Its neurons have diverse genetic profiles and influence a variety of homeostatic functions. While its cytoarchitecture and overall efferent projections are known, we lack comprehensive information on the projection patterns of specific neuronal subtypes in the PB. In this study, we compared the projection patterns of glutamatergic neurons here with a subpopulation expressing the transcription factor Foxp2 and a further subpopulation expressing the neuropeptide Pdyn. To do this, we injected an AAV into the PB region to deliver a Cre-dependent anterograde tracer (synaptophysin-mCherry) in three different strains of Cre-driver mice. We then analyzed 147 neuroanatomical regions for labeled boutons in every brain (n = 11). Overall, glutamatergic neurons in the PB region project to a wide variety of sites in the cerebral cortex, basal forebrain, bed nucleus of the stria terminalis, amygdala, diencephalon, and brainstem. Foxp2 and Pdyn subpopulations project heavily to the hypothalamus, but not to the cortex, basal forebrain, or amygdala. Among the few differences between Foxp2 and Pdyn cases was a notable lack of Pdyn projections to the ventromedial hypothalamic nucleus. Our results indicate that genetic identity determines connectivity (and therefore, function), providing a framework for mapping all PB output projections based on the genetic identity of its neurons. Using genetic markers to systematically classify PB neurons and their efferent projections will enhance the translation of research findings from experimental animals to humans., (© 2020 Wiley Periodicals LLC.)

Obesity is associated with an impaired balance of CD4 + T cell subsets. Both vitamin D and obesity have been reported to affect the mTOR pathway. In this study, we investigated the effects of vitamin D on CD4 + T cell subsets and the mTOR pathway. Ten-week-old male C57BL/6 mice were divided into four groups and fed diets with different fat (control or high-fat diets: CON or HFD) and vitamin D contents (vitamin D control or supplemented diets: vDC or vDS) for 12 weeks. T cells purified by negative selection were stimulated with anti-CD3/anti-CD28 mAbs and cultured for 48 h. The percentage of CD4 + IL-17 + T cells was higher in the vDS than vDC groups. The CD4 + CD25 + Foxp3 + T cells percentage was higher in HFD than CON groups. The phospho-p70S6K/total-p70S6K ratio was lower in vDS than vDC, but the phospho-AKT/total-AKT ratio was higher in vDS than vDC groups. Hif1α mRNA levels were lower in vDS than vDC groups. These findings suggest HIF1α plays an important role in vitamin-D-mediated regulation of glucose metabolism in T cells, and dietary vitamin D supplementation may contribute to the maintenance of immune homeostasis by regulating the mTOR pathway in T cells., Competing Interests: The authors declare no conflict of interest.

Backgroundː Chronic kidney disease stage G5 (CKD G5) patients show an activated but impaired immune system. One function of the FOXP3 + regulatory T (Treg) cells is to preserve tolerance to self while maintaining the ability to fight infectious agents. The aim of this pilot study is to evaluate longitudinal changes in Treg cells before and 1 month after the first dialysis treatment. Materials and methodsː CKD G5 patients not yet on dialysis were enrolled and started on hemodialysis (HD) or peritoneal dialysis (PD). Tregs were analyzed by flow cytometry at two time points: T0 (before the first dialysis treatment) and T1 (1 month after the first dialysis session). Wilcoxon test for dependent samples was used to compare the mean percentage difference between T0 and T1: Δ% = 100 × [(T1 - T0) / T0]. Resultsː 21 patients were enrolled: 8 on HD and 13 on PD. The proportion of total lymphocytes (low side scatter lymphocyte gate) and T lymphocytes (in the CD3 + CD4 + gate) did not change significantly 1 month after the start of dialysis in both groups. Treg cells (as CD25 + FOXP3 + , FOXP3 + , or CD25 + CD127 - ), analyzed as percentage of the lymphocyte gate, showed a significant increase post PD (CD25 + FOXP3 + : median = 35.92; p = 0.0425; FOXP3 + : median = 30.85; p = 0.0479 and CD25 + CD127 - : median = 23.71; p = 0.0215). The same populations, did not change 1 month after the first dialysis session. Conclusionː Our study is the first to evaluate longitudinal effects of dialysis on Treg cells in uremia and suggests that PD was more effective in increasing Treg levels 1 month post initiation of dialysis and may contribute to improvement of inflammatory status. Thus, PD may contribute to better outcomes for patients with renal dysfunction, also maintaining homeostasis of peritoneal and renal tissues.

Objectives: The impact of regulatory T (Treg) cells as a prognostic factor of survival in head and neck squamous cell carcinoma (HNSCC) remains controversial. We aimed to evaluate the prognostic value of Treg cells in patients with HNSCC through a meta-analysis., Materials and Methods: Through a literature search in PubMed, Embase, and Cochrane, we included 11 articles in this meta-analysis and investigated the effect of Treg cell level on the survival of patients with HNSCC. Also, we performed a subgroup analysis according to the study sample (blood vs. tumor tissue), primary tumor site, HPV infectivity, or Treg cell marker., Results: High levels of circulating Treg cells in the peripheral blood of patients with HNSCC can significantly increase the disease specific survival rate of patients. Moreover, subgroup analysis showed that high levels of Treg in peripheral blood were significantly associated with better disease specific survival in patients with oral cancer, a subsite of HNSCC, but not in those with other head and neck subsite. Positivity of HPV infection did not influence the prognosis of patients with HNSCC., Conclusion: Increase in the levels of circulating Treg cells in peripheral blood can be a prognostic factor of survival in patients with oral cancer., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Anorectal malignant melanoma (ARMM) is a rare disease with poor prognosis. Determining ARMM prognosis precisely is difficult due to the lack of proper assessment techniques. Immunotherapy has proven effective against cutaneous malignant melanoma and may show efficacy in ARMM. Herein, we assessed the immune profile of ARMM to identify possible prognostic biomarkers. Twenty-two ARMM formalin-fixed and paraffin-embedded samples were evaluated using an nCounter ® PanCancer Immune Profiling Panel. Validation was performed through immunohistochemical staining for CD3, CD8, Foxp3, CD68, CD163, and PD-L1. RNA analysis revealed significantly decreased scores for pathways involved in cell regulation and function, as well as chemokines, in recurrent patients compared to nonrecurrent patients. In cell-type profiling, the recurrent cases displayed significantly low tumor infiltrating lymphocyte (TIL) scores. Recurrence/death prediction models were defined using logistic regression and showed significantly lower scores in recurrent and deceased patients (all, P < 0.001) compared to those in nonrecurrent and surviving patients. The high total TIL and tumor-associated macrophage (TAM) groups had significantly better overall survival outcomes compared to the low total TIL and TAM groups (P = 0.007 and P = 0.035, respectively). In addition, the presence of CD3 + TILs in the invasion front was an independent favorable prognostic indicator (P = 0.003, hazard ratio = 0.21, 95% confidential interval, 0.01-0.41). Patients with inflamed or brisk-infiltration type tumors also had a significantly better overall survival than that of patients with immune-desert/excluded and absent/non-brisk type tumors (P = 0.03 and P = 0.0023, respectively). In conclusion, TILs have a strong prognostic value in ARMM, and the quantification of TILs and an analysis of the TIL phenotype and infiltration pattern during pathological diagnosis are essential to guide treatment strategies and accurate prognosis in ARMM.

Background/aim: Upregulation of Forkhead box G1 (FOXG1) has recently been observed in many cancers, while its effect on radiosensitivity in glioma is still unclear. In this study, we hypothesized that FOXG1 be a major player in radioresistance of glioma as well as the underlying mechanism., Methods: Immunohistochemistry (IHC) was conducted to assess FOXG1 expression in glioma tissues and glioma-adjacent tissues. Western Blot was implemented to detect the expression of autophagy-related proteins. CCK-8, colony formation and flow cytometry assays were implemented to assess cell viability, proliferation and apoptosis, respectively. Transmission electron microscope (TEM) was used to observe autophagic vesicles. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay was applied to detect the expression of FOXG1., Results: The present study demonstrated that FOXG1 was highly expressed in glioma tissues. FOXG1 expression level was up-regulated in glioma cells following exposure to X-ray irradiation. FOXG1 can attenuate radiosensitivity of glioma cells. Moreover, it revealed that FOXG1 attenuate radiosensitivity of glioma cells by promoting autophagy., Conclusions: The present study suggests that FOXG1 is a pivotal molecule for circumventing radiation-induced cell death in malignant glioma cells through the regulation of autophagy, and it may be a target for the treatment of human brain glioma.

Objective: To investigate the correlations of Helicobacter pylori (HP) with liver function, inflammatory factors and serum levels of forkhead box P3 (FoxP3) and retinoic acid receptor-related orphan receptor gamma-t (RORγt) in patients with hepatitis B cirrhosis (HBC)., Patients and Methods: A total of 60 HBC patients were divided into HBC group (n=30) and HP-infected HBC group (HP&HBC group, n=30). QRT-PCR was conducted to determine the messenger ribonucleic acid (mRNA) levels of FoxP3 and RORγt in serum samples. ELISA was applied to measure the levels of relevant inflammatory factors. Besides, immunohistochemical staining was conducted to detect positive expressions of FoxP3 and RORγt in liver tissues of patients in the two groups., Results: No significant differences in gender, drinking, smoking, diabetes and age were found between HBC group and HP&HBC group (p>0.05). Globulin and albumin levels were comparable between the two groups (p>0.05). Liver function indexes, including ALT, AST and TBIL were higher in HP&HBC group than those in HBC group (p<0.05). The HBV-DNA level was lower in HBC group in comparison with that in HP&HBC group. The interferon-gamma (IFN-γ) level was remarkably higher in HBC group than that in HP&HBC group (p<0.01), and the levels of interleukin (IL)-6, IL-10, IL-17 and transforming growth factor (TGF)-β1 were notably lower in HBC group in comparison with those in HP&HBC group (p<0.01). Additionally, the mRNA levels of FoxP3 and RORγt in HBC group were distinctly lower than those in HP&HBC group (p<0.01). The mRNA levels of FoxP3 and RORγt were positively related to those of IL-6, IL-10, IL-17, and TGF-β1, and negatively associated with IFN-γ level. Immunohistochemical results indicated that positive expression rates of FoxP3 and RORγt in the liver tissues were approximately 50% in HP&HBC group and B. Zhao, Q.-J. Sheng, Y. Qin, X.-L. Wang, H. Zhao, N. Zhaowere 15% in HBC group, and the difference was statistically significant (p<0.05)., Conclusions: Expression levels of FoxP3 and RORγt in serum and liver tissues are elevated in HP-infected HBC patients, and inflammatory factors are correlated with their expressions, suggesting the aggravated liver damage.

Background: Differences in the expression of regulatory T cells (Tregs) have been suggested to explain why some smokers develop COPD and some do not. Upregulation of Tregs in response to smoking would restrain airway inflammation and thus the development of COPD; while the absense of such upregulation would over time lead to chronic inflammation and COPD. We hypothesized that-among COPD patients-the same mechanism would affect rate of decline in lung function; specifically, that a decreased expression of Tregs would be associated with a more rapid decline in FEV 1 ., Methods: Bronchoscopy with BAL was performed in 52 subjects recruited from the longitudinal OLIN COPD study; 12 with COPD and a rapid decline in lung function (loss of FEV 1  ≥ 60 ml/year), 10 with COPD and a non-rapid decline in lung function (loss of FEV 1  ≤ 30 ml/year), 15 current and ex-smokers and 15 non-smokers with normal lung function. BAL lymphocyte subsets were determined using flow cytometry., Results: The proportions of Tregs with regulatory function (FoxP3 + /CD4 + CD25 bright ) were significantly lower in COPD subjects with a rapid decline in lung function compared to those with a non-rapid decline (p = 0.019). This result was confirmed in a mixed model regression analysis in which adjustments for inhaled corticosteroid usage, smoking, sex and age were evaluated. No significant difference was found between COPD subjects and smokers or non-smokers with normal lung function., Conclusions: COPD subjects with a rapid decline in lung function had lower proportions of T cells with regulatory function in BAL fluid, suggesting that an inability to suppress the inflammatory response following smoking might lead to a more rapid decline in FEV 1 . Trial registration Clinicaltrials.gov identifier NCT02729220.

Ovarian high-grade serous carcinomas (HGSC) have shown lackluster responses to immunotherapies targeting the PD-1/PD-L1 axis, perhaps due to the coexistence of other mechanisms of immune evasion in this tumor type. Lymphocyte activation gene-3 (LAG-3) is another inhibitory immune checkpoint often expressed on tumor-associated lymphocytes which is targeted by drugs currently in clinical trials. Forty-eight HGSC with known germline BRCA mutation status were immunohistochemically stained for LAG-3, CD8, and FOXP3. Positive tumor-associated lymphocytes were enumerated and averaged over 10 high-power fields (HPF). PD-L1 immunostaining was also preformed and expression was evaluated on tumor cells and using the combined positive score (CPS). The average number of LAG-3-positve tumor-associated lymphocytes was 6/HPF (range: 0-25.6). Cytotoxic (CD8) T cells averaged 30/HPF (range: 0-168.9), and regulatory (FOXP3) cells averaged 6.6/HPF (range: 0-76.3). Tumoral PD-L1 expression of ≥1% was observed in 27% (13/48) of cases, with only 8% (4/48) showing >5% staining; 81% (39/48) cases had a CPS ≥1. LAG-3-positive lymphocytes and PD-L1 expression were positively correlated, even after controlling for the overall level of CD8 and FOX3P lymphocyte infiltration. Germline BRCA status was not significantly associated with LAG-3, CD8, FOXP3, or PD-L1 expression. These findings indicate that immunotherapies targeting LAG-3 may benefit some ovarian HGSC patients, particularly when used in conjunction with anti-PD-1/PD-L1 approaches. The typically limited expression of LAG-3 and PD-L1 suggests that immunotherapeutic response may be muted in most HGSC even with a combination approach.

Purpose: Forkhead box C1 (FOXC1) is critical for maintaining bone marrow microenvironments during hematopoiesis, but its role in hematological malignancies remains obscure. Here, we investigated whether FOXC1 regulates tumor dormancy and activation in the microenvironments of T and natural killer (NK) cell lymphomas., Materials and Methods: One hundred and twenty cases of T and NK cell lymphomas were included; the immunohistochemical expression of FOXC1 was investigated in stromal cells, and numbers of FOXC1+ stromal cells were counted. Furthermore, the expression of phosphorylated p38 (p-p38) and phosphorylated ERK1/2 (p-ERK1/2) in tumor cells was investigated using immunohistochemistry., Results: FOXC1 was variably expressed in C-X-C motif chemokine 12-associated reticular stromal cells, histiocytes, (myo)fibroblasts, and endothelial cells. The phenotypes of cases were categorized as dormant (high p-p38/low p-ERK1/2; n=30, 25.0%), active (high p-ERK1/2/low p-p38; n=25, 20.8%), or intermediate (others; n=65, 54.2%). Lower FOXC1+ stromal cell infiltration was associated with the dormant phenotype, the precursor T lymphoblastic leukemia/lymphoma subtype, and inferior overall survival rates, whereas higher FOXC1+ stromal cell infiltration was associated with the active phenotype and favorable patient prognosis (p < 0.05 for all)., Conclusion: These results suggested that FOXC1+ stromal cells within the microenvironments of T and NK cell lymphomas might be related to tumor phenotypes.

Background: Tumour infiltrating lymphocytes (TILs) are an established prognostic biomarker for triple-negative breast cancer (TNBC). We evaluated the role of programmed cell-death ligand-1 (PD-L1), CD8 and FOXP3 expression in refining a prognostic model for non-metastatic TNBC beyond classic factors and TILs., Methods: Primary tumour samples from 244 early patients with TNBC, all treated with surgery and chemotherapy, were collected. Stromal TILs were evaluated on haematoxylin-eosin slides according to guidelines. PD-L1, CD8 and FOXP3 were assessed by immunohistochemistry and evaluated by digital pathology., Results: TILs, PD-L1, CD8 and FOXP3 were positively correlated with each other (P < 0.001). TILs were confirmed as an independent prognostic factor. When PD-L1, CD8 and FOXP3 were added to multivariable models including classic factors (age, stage, histologic grade) and TILs, PD-L1 provided the largest amount of additional prognostic information: likelihood ratio χ 2 4.60, P = 0.032 (in a model including classic factors and TILs 10% increments) and likelihood ratio χ 2 6.50, P = 0.011 (in a model including classic factors and TILs >30% versus <30%). In the subset of patients treated with neoadjuvant chemotherapy, FOXP3 provided further prognostic information beyond classic factors, TILs and pathological complete response (pCR) (likelihood ratio χ 2 5.01, P = 0.025). For patients who did not achieve a pCR, the expression of CD8 and PD-L1 was significantly increased from baseline to residual disease., Conclusions: Beyond clinicopathological factors and TILs, other immune biomarkers may add prognostic information for early TNBC. The increased PD-L1 expression on residual disease after neoadjuvant chemotherapy strengthens the rationale of testing immune checkpoint inhibitors in the post-neoadjuvant setting., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Purpose: To evaluate radiotherapy-induced changes in the expression of programmed death ligand 1 (PD-L1), programmed death 1 (PD-1), and human leukocyte antigen class I (HLA-1) in patients with uterine cervical cancer, as well as infiltration of CD8+ and Forkhead box P3+ (FoxP3+) T lymphocytes into tumor tissue and the prognostic value of these parameters., Materials and Methods: We performed immunohistochemical analysis of pre-radiotherapy biopsies and corresponding post-radiotherapy resected tissues in 104 uterine cervical cancer patients undergoing preoperative chemoradiotherapy or radiotherapy alone. We scored the expression of various proteins to distinguish positive from negative samples., Results: PD-L1-expressing tumor cells (PD-L1 TC) increased significantly after chemoradiotherapy (p = 0.043). CD8+ T cell infiltration (p = 0.002) and FoxP3+ T cell infiltration (p = 0.003) decreased significantly after chemoradiotherapy. Expression of PD‑1, PD-L1-expressing immune cells (PD-L1 IC), and HLA‑1 did not change after chemoradiotherapy. In biopsy specimens obtained before chemoradiotherapy or radiotherapy, greater infiltration of CD8+ T cells (p = 0.001) and FoxP3+ T cells (p = 0.003) were significant predictors of better overall survival (OS). In surgical specimens obtained after chemoradiotherapy or radiotherapy, greater infiltration of PD-L1 TC was the only significant predictor of better OS (p < 0.001) and was related to a significantly lower probability of out-of-field recurrence (p = 0.005)., Conclusion: Chemoradiotherapy induced an immunologic shift that increased PD-L1 TC. Chemoradiotherapy has immunological effects that can influence the results of treatment for uterine cervical cancer.

Background: Radiotherapy is a standard of care for locally advanced stage III N2 non-small-cell lung carcinoma (NSCLC) combined with surgery/chemotherapy. Radiotherapy is hypothesised to induce tumour immunogenic cell death, to release neoantigen resulting in intra-tumoural immune infiltration and abscopal effect. Conversely, it has not been demonstrated if immune cells are necessary to drive radiotherapy efficacy and predict patient's survival., Patients and Methods: We retrospectively analysed tumour samples and clinical data from 113 patients, 89 resected (PORT) and 24 non-resected (DRC) N2-NSCLC treated with chemotherapy and radiotherapy (same radiotherapy department from 2002 to 2015). The immune environment was characterised with in situ multiplex staining (CD8, FoxP3, PD-L1 and cytokeratin) and correlated with clinical data and survival., Results: High density of CD8+ T cells was associated with OS (p = 0.04, HR = 1.93 [0.99-3.78]) and DFS (p = 0.003, HR = 2.42 [1.31-4.47]) in the PORT. High density of CD8+/FoxP3+ double positive cells was associated with OS (p = 0.01, HR = 1.97 [1.11-3.48]) in the whole population, with OS (p = 0.05, HR = 1.92 [0.98-3.74]) and PFS (p = 0.03, HR = 1.83 [1.03-3.23]) in the PORT without reaching significance for the DRC. Intermediate PD-L1 expression in tumour cells (TPS = 1-49%) was associated with a higher survival in the PORT., Conclusions: Intra-tumoural CD8+ T cell and particularly CD8+/FoxP3+ double positive T cell densities predict survival in stage III N2-NSCLC suggesting the need for a pre-existing intra-tumour immunity to mediate the action of radiotherapy. Density of CD8+/FoxP3+ cells was the best predictor of patient's survival in multivariate analysis and could represent a biomarker of radiotherapy efficacy., Competing Interests: Conflict of interest statement GB: Received support from the SFRO. ALM: Lectures and educational activities: AstraZeneca, Roche, Pfizer. Travel, accommodations, expenses: Roche. HB: Lectures and educational activities: AstraZeneca, Bristol-Myers Squibb, MSD. MW: Lectures and educational activities: Astra-Zeneca, Amgen, Boehringer Ingelheim, Bristol-Myers Squibb, MSD, Roche. Travel, accommodations, expenses: Astra-Zeneca, MSD, Roche, Pfizer. JT: Travel, accommodations, expenses: BMS, Roche. SAH and RH: Full-time employees of AstraZeneca. MA: Consulting advisory role: AstraZeneca. PG: Lectures and educational activities: Astra-Zeneca, Bristol-Myers Squibb, Ipsen, Merck. DD: Lectures and educational activities: AstraZeneca, Bristol-Myers Squibb, MSD, Agilent. Consulting advisory role: AstraZeneca, BMS, MSD. Travel, accommodations, expenses: Abbvie, Roche, AstraZeneca. Sponsored research: AstraZeneca, Medimmune, Roche. The remaining authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: Breast cancer tumour-infiltrating lymphocytes associate with clinico-pathological factors, including survival, although the literature includes many conflicting findings. Our aim was to assess these associations for key lymphocyte subtypes and in different tumour compartments, to determine whether these provide differential correlations and could, therefore, explain published inconsistencies. Uniquely, we also examine whether infiltrating levels merely reflect systemic lymphocyte levels or whether local factors are predominant in recruitment., Methods: Immunohistochemistry was used to detect tumour-infiltrating CD20+ (B), CD4+ (helper T), CD8+ (cytotoxic T) and FoxP3+ (regulatory T) cells in breast cancers from 62 patients, with quantification in tumour stroma, tumour cell nests, and tumour margins. Levels were analysed with respect to clinico-pathological characteristics and matched circulating levels (determined by flow-cytometry)., Results: CD4+ lymphocytes were the most prevalent subtype in tumour stroma and at tumour edge and CD8+ lymphocytes were most prevalent in tumour nests; FoxP3+ lymphocytes were rarest in all compartments. High grade or hormone receptor negative tumours generally had significantly increased lymphocytes, especially in tumour stroma. Only intra-tumoural levels of CD8+ lymphocytes correlated significantly with matched circulating levels (p < 0.03), suggesting that recruitment is mainly unrelated to systemic activity. High levels of stromal CD4+ and CD20+ cells associated with improved survival in hormone receptor negative cases (p < 0.04), while tumour nest CD8+ and FoxP3+ cells associated with poor survival in hormone receptor positives (p < 0.005)., Conclusions: Lymphocyte subtype and location define differential impacts on tumour biology, therefore, roles of tumour-infiltrating lymphocytes will only be unravelled through thorough analyses that take this into account.

Background: CXCL4 plays an essential role in the regulation of multiple immune diseases. However, the underlying role of CXCL4 is still not clear in sepsis. Aim: In the present study, we aimed to investigate the function of CXCL4 in sepsis. Methods: Sepsis model was constructed on mouse. Flow cytometry was used to determine the ratio of CD4 + CD25 + FOXP3 + Treg cells. ELISA assays were used to determine the levels of CXCL4, IL-6, IL-10, and TNF-α respectively. Western blot was used to examine protein contents. Results: Our results suggested that the serum level of CXCL4 was upregulated in patients with sepsis and positively associated with the ratio of human CD4 + CD25 + FOXP3 + Treg cells. To further examine the role of CXCL4 in sepsis, we constructed the mouse sepsis model. Our results indicated that the mouse antibody of CXCL4 treatment reduced the expression of urine creatinine and urea nitrogen in sepsis model. Moreover, the frequency of CD25 + FOXP3 + mouse regulatory T cells (Tregs) cells was decreased in mouse CD4 + T cells in the presence of mouse CXCL4 antibody. Further, the mouse recombinant protein CXCL4 was used to culture normal mouse CD4 + T cells in vitro . Our finding indicated that the recombinant protein CXCL4 promoted the percentage of mouse CD25 + FOXP3 + Treg cells and enhanced the phosphorylation of STAT5 in mouse CD4 + T cells in a dose-dependent manner. However, these effects were significantly reversed by the STAT5 inhibitor ( p  < .001). Conclusion: our findings not only indicated the function and signalling pathway of CXCL4 in CD4 + T cells but also provided novel insight and target in sepsis treatment.

We investigated whether a unique immune response was instigated with the development of oral tongue squamous cell carcinomas (OTSCC), with/without nodal involvement, with/without recurrent metastatic disease, or within tumor involved nodes. One hundred and ten formalin-fixed paraffin-embedded samples were collected from a retrospective cohort of 67 OTSCC patients and 10 non-cancerous tongue samples. Targets including CD4, CD8, FOXP3, PD-L1, and PD-1 were analyzed by immunohistochemistry. The Nanostring PanCancer Immune Profiling Panel was used for gene expression profiling. Data were externally validated in the The Cancer Genome Atlas (TCGA) head and neck (HNSCC), melanoma and lung squamous cell carcinoma (LSCC) cohorts. A 24-immune gene signature was identified that discriminated more aggressive OTSCC cases, and although not prognostic in HNSCC was associated with survival in other TCGA cohorts (improved survival for melanoma, P < .001 and worse survival for LSCC, P = .038). OTSCC exhibited concordant gene and immunohistochemical (IHC) features characterized by a TH-2 biased, proinflammatory profile with upregulated B cell and neutrophil gene activity and increased CD4, FOXP3, and PD-L1 expression (P < .001 for all by IHC). Compared to less advanced disease, nodal involvement and recurrent OTSCC did not induce a different immune response although recurrent disease was characterized by significantly higher PD-L1 expression (P = .004 by SP263, P = .013 by 22C3, P = .004 for gene expression). Identification of a gene signature associated with different prognostic effects in other cancers highlights common pathways of immune dysregulation that are impacted by the tumor origin. The significant immunosuppressive signaling in OTSCC indicates primary failure of immune system to control carcinogenesis emphasizing the need for early, combination therapeutic approaches., (© 2020 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)