CD4+CD69+ T cells and CD4+CD25+FoxP3+ Treg cells imbalance in peripheral blood, spleen and peritoneal lavage from pristane-induced systemic lupus erythematosus (SLE) mice

Abstract Background: Adaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model. Methods: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant. Results: Compared with the controls, SLE-induced animals presented increased numbers of CD4+CD69+ T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4+ CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFβ1 (p = 0.038). Conclusion: Increased numbers of CD4+CD69+ T cells and reduced numbers of CD4+CD25+FoxP3+ Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.

Immunohistochemical evaluation of HLA-G and FoxP3+ T regulatory cells in oral cavity and lower lip squamous cell carcinomas

Abstract: Human Leukocyte Antigen G (HLA-G) is a molecule involved in the tumor immunosuppression and also in the generation of regulatory T (Treg) cells, thus leading to evasion to the immune system host, and consequently, contributing to tumor progression in several cancers. The aim of this study was to evaluate the immunoexpression of HLA-G by tumor cells and FoxP3+ Treg cells in 25 oral tongue squamous cell carcinomas (SCCs) and 25 lower lip SCCs and analyze their relationship with clinical parameters. HLA-G expression was higher in oral tongue SCCs than in lower lip SCCs. In oral tongue SCCs and lower lip SCCs, no association between HLA-G expression and clinical parameters (tumor size, lymph node status, distant metastasis, and clinical stage) was verified (P>0.05). FoxP3+ Treg cells were detected along the tumor invasive front in all cases of oral tongue and lower lip SCCs. In oral tongue SCC cases, the number of Treg cells tended to be higher in smaller tumors, tumors without regional lymph node metastasis, and tumors in early clinical stages, but the difference was not statistically significant (P>0.05). A significant positive correlation was found between the expression of HLA-G by neoplastic cells and Treg cells in lower lip SCCs (p = 0.008). Our findings suggest the involvement of HLA-G and Treg cells in the modulation of immune responses in oral tongue and lower lip SCCs. This interaction between HLA-G and Treg cells may represent an evasion mechanism in these malignancies.

Treg and Th17 cells in inflammatory periapical disease: a systematic review

Abstract: The process involved in periapical lesions, which occur as an outcome of pulpal necrosis, is regulated by the immune system including regulatory T cells (Treg) and T helper 17 cell (Th17) responses. The objective of this study was to conduct a frequency systematic review to determine the presence of Treg/Th17 responses and the influence of these cells in the progression of chronic inflammatory periapical lesions in humans. A systematic computerized search was carried out in Pubmed, Medline, Web of Science and Scopus electronic databases from their date of inception through the first week of May 2017. In addition, the reference lists of the included articles and the grey literature were hand-searched. Articles that evaluated the presence and influence of Treg/Th17 in the progression of human periapical lesions were included. Study selection and the quality assessment of the included articles (using the Newcastle-Ottawa scale) were carried out by two authors. Fifty-seven titles/abstracts were screened and eight studies met the eligibility criteria and were included in this systematic review. The included studies showed large variation in the type of periapical lesion assessed, mean age, age range, type of experiment and findings regarding the participation of Th17 and Treg in the status of inflammatory periapical lesions. The studies showed the involvement of Treg in the modulation of the inflammatory response in radicular cysts and periapical granulomas. This systematic review highlights the relationship between Treg and Th17 acting in a subtle balance inhibiting or promoting the progression of human periapical lesions.

Immunohistochemical analysis of FoxP3+ regulatory T cells in lower lip squamous cell carcinomas

Abstract: The aim of this study was to determine the number of FoxP3+ regulatory T (Treg) cells in the microenvironment of lower lip squamous cell carcinomas (LLSCCs) and to correlate the findings with clinicopathological parameters (tumor size/extent, regional lymph node metastasis, clinical stage, and histopathological grade of malignancy). Fifty cases of LLSCC were selected. Lymphocytes exhibiting nuclear immunostaining for FoxP3 were quantified in 10 microscopic fields at the deep invasive front of LLSCCs. The results were analyzed statistically using the nonparametric Mann-Whitney test and Fisher's exact test. FoxP3+ lymphocytes were observed in all cases studied. The number of these cells tended to be higher in smaller tumors, tumors without regional lymph node metastasis, and tumors in early clinical stages, but the difference was not statistically significant (p > 0.05). Low-grade tumors contained a larger number of FoxP3+ lymphocytes than high-grade tumors (p = 0.019). Tumors with an intense inflammatory infiltrate exhibited a larger number of Treg cells (p = 0.035). On the other hand, the number of FoxP3+ lymphocytes was smaller in tumors arranged in small cell clusters (p = 0.003). No significant differences in the number of FoxP3+ lymphocytes were observed according to the degree of keratinization (p = 0.525) or nuclear pleomorphism (p = 0.343). The results suggest the participation of Treg cells in immune and inflammatory responses in the microenvironment of LLSCCs. These cells may play a more important role in early stages rather than in advanced stages of lip carcinogenesis.

T regulatory cells (TREG)(TCD4+CD25+FOXP3+) distribution in the different clinical forms of leprosy and reactional states

BACKGROUND: Leprosy is characterized histologically by a spectrum of different granulomatous skin lesions, reflecting patients' immune responses to Mycobacterium leprae. Although CD4+CD25+ FoxP3+ T regulatory cells are pivotal in the immuneregulation, presence, frequency, and distribution of Tregs in leprosy, its reactional states have been investigated in few studies. OBJECTIVES: This study aimed to verify the frequency and distribution of regulatory T cells in different clinical forms and reactional states of leprosy. METHODS: We performed an immunohistochemical study on 96 leprosy cases [Indeterminate (I): 9 patients; tuberculoid tuberculoid: 13 patients; borderline tuberculoid: 26 patients; borderline borderline: 3 patients; borderline lepromatous: 8 patients; lepromatous lepromatous: 27 patients; reversal reaction: 8 patients; and erythema nodosum leprosum: 2 patients]. RESULTS: FoxP3-positive cells were present in 100% of the cases with an average density of 2.82% of the infiltrate. Their distribution was not related to granulomatous structures or special locations. There was a statistically significant increment of FoxP3 expression in patients with leprosy reversal reactions when compared with patients presenting with type I leprosy (P= 0.0228); borderline tuberculoid leprosy (P = 0.0351) and lepromatous leprosy (P = 0.0344). CONCLUSIONS: These findings suggest that Tregs play a relevant role in the etiopathogenesis of leprosy, mainly in type I leprosy reaction. .

Increased Expression of Forkhead Box M1 Is Associated with Aggressive Phenotype and Poor Prognosis in Estrogen Receptor-Positive Breast Cancer

Fox transcription factors play a critical role in the regulation of a variety of biological processes. While FoxM1 behaves like the oncogenic transcription factor, FoxO3a is known as a tumor suppressor by inhibiting FoxM1. This study aimed to investigate the clinicopathological significance of FoxM1 and FoxO3a expression in breast cancer. Expression of FoxM1 and FoxO3a were analyzed by immunohistochemical staining on tissue microarray sections from 236 breast cancer patients, and correlated with various clinicopathological characteristics. Overexpression of FoxM1 correlated with adverse clinicopathological features, such as larger tumor size, lymph node metastasis, advanced tumor stage, and lymphovascular invasion. The Kaplan-Meier survival curves revealed no prognostic significance of FoxM1 expression. However, in subgroup analyses with patients of estrogen receptor (ER) positive breast cancers, FoxM1 overexpression associated with poor disease free and overall survival. No association was found between FoxO3a and FoxM1 expression. Regarding clinicopathological variables, the only association between histologic grade and FoxO3a was observed. In conclusion, FoxM1 overexpression was significantly associated with aggressive phenotypes and poor prognosis of ER-positive breast cancer. These findings suggest the possible role of FoxM1 as a prognostic biomarker and putative target of anti-cancer therapy.

Imbalanced expression of functional surface molecules in regulatory and effector T cells in systemic lupus erythematosus

Regulatory T (TREG) cells play an important role in maintaining immune tolerance and avoiding autoimmunity. We analyzed the expression of membrane molecules in TREG and effector T cells in systemic lupus erythematosus (SLE). TREG and effector T cells were analyzed for the expression of CTLA-4, PD1, CD28, CD95, GITR, HLA-DR, OX40, CD40L, and CD45RO in 26 patients with active disease, 31 with inactive disease, and 26 healthy controls. TREG cells were defined as CD25+/highCD127Ø/lowFoxP3+, and effector T cells were defined as CD25+CD127+FoxP3Ø. The ratio of TREG to effector T cells expressing GITR, PD1, HLA-DR, OX40, CD40L, and CD45RO was determined in the three groups. The frequency of TREG cells was similar in patients with SLE and controls. However, SLE patients had a decreased frequency of CTLA-4+TREG and CD28+TREG cells and an increased frequency of CD40L+TREG cells. There was a decrease in the TREG/effector-T ratio for GITR+, HLA-DR+, OX40+, and CD45RO+ cells, and an increased ratio of TREG/effector-T CD40L+ cells in patients with SLE. In addition, CD40L+TREG cell frequency correlated with the SLE disease activity index (P=0.0163). In conclusion, our findings showed several abnormalities in the expression of functionally critical surface molecules in TREG and effector T cells in SLE that may be relevant to the pathogenesis of this disease.

Thymopoiesis and regulatory T cells in healthy children and adolescents

OBJECTIVES: The purpose of this study was to investigate the association between T cell receptor excision circle levels in peripheral blood mononuclear cells and regulatory T cells that co-express CD25 and Foxp3 in healthy children and adolescents of different ages. MATERIALS AND METHODS: The quantification of signal-joint T-cell receptor excision circle levels in the genomic DNA of peripheral blood mononuclear cells was performed using real-time quantitative PCR. The analysis of CD4, CD8, CD25, and Foxp3 expression was performed using flow cytometry. RESULTS: Ninety-five healthy controls (46 females and 49 males) ranging in age from 1 to 18 years were analyzed. The mean T-cell receptor excision circle count in all individuals was 89.095¡36.790 T-cell receptor excision circles per microgram of DNA. There was an inverse correlation between T-cell receptor excision circles counts and age (r = -0.846; p<0.001) as well as between the proportion of CD4+CD25+Foxp3+ T cells and age (r = -0.467; p = 0.04). In addition, we observed a positive correlation between the amount of CD4+CD25+Foxp3+ T cells and the amount of Tcell receptor excision circles per microgram of DNA in individuals of all ages (r = -0.529; p = 0.02). CONCLUSIONS: In this study, we observed a decrease in the thymic function with age based on the fact that the level of T-cell receptor excision circles in the peripheral blood positively correlated with the proportion of regulatory T cells in healthy children and adolescents. These findings indicate that although T-cell receptor excision circles and regulatory T cells levels decrease with age, homeostasis of the immune system and relative regulatory T cells population levels are maintained in the peripheral blood.

Foxp3 expression is associated with aggressiveness in differentiated thyroid carcinomas

OBJECTIVES: Forkhead box P3 (FoxP3) expression has been observed in human cancer cells but has not yet been reported in thyroid cells. We investigated the prognostic significance of both FoxP3 expression and intratumoral FoxP3+ lymphocyte infiltration in differentiated thyroid carcinoma cells. METHODS: We constructed a tissue microarray with 385 thyroid tissues, including 266 malignant tissues (from 253 papillary thyroid carcinomas and 13 follicular carcinomas), 114 benign lesions, and 5 normal thyroid tissues. RESULTS: We determined the expression of FoxP3 in both tumor cells and tumor-infiltrating lymphocytes using immunohistochemical techniques. Cellular expression of FoxP3 was evident in 71% of benign and 91.9% of malignant tissues. The nuclear and cytoplasmic expression patterns were quantified separately. A multivariate logistic regression analysis indicated that cytoplasmic FoxP3 expression is an independent risk factor for thyroid malignancy. Cytoplasmic FoxP3 staining was inversely correlated with patient age. Nuclear FoxP3 staining was more intense in younger patients and in tumors presenting with metastasis at diagnosis. FoxP3+ lymphocytes were more frequent in tumors smaller than 2 cm, those without extrathyroidal invasion, and in patients with concurrent chronic lymphocytic thyroiditis. CONCLUSIONS: We demonstrated FoxP3 expression in differentiated thyroid carcinoma cells and found evidence that this expression may exert an important influence on several features of tumor aggressiveness.

Expansion of peripheral and intratumoral regulatory T-cells in hepatocellular carcinoma: A case-control study.

Background: Hepatocellular carcinoma (HCC) is notorious for poor prognosis with limited therapeutic options. A better understanding of the role of regulatory T-cells (Tregs) in HCC is important for design of immunotherapy based clinical protocol. The objective of the present study was to evaluate the presence of Tregs in tumor microenvironment in patients with HCC compared to chronic hepatitis (CH). Materials and Methods: The frequency of CD4 + CD25 + Treg cells was evaluated from peripheral blood (PB) of 28 patients of HCC and 30 controls including CH cases and healthy donors using flowcytometry. Intratumoral Treg were also analyzed in tissue samples from 17 HCC cases and 15 CH cases. In addition the expression of FOXP3 and CTLA-4 was also studied by RT-PCR. Results: Frequency of CD4 + CD25 + cells in the PBMCs of HCC cases was significantly higher than in HC (10.8 ± 7.64 vs 3.05 ± 1.30, P < 0.005) and CH patients (2.88 ± 1.92, P < 0.005). Also Treg population was significantly higher in HCC tumor microenvironment compared to CH biopsies (15.8 ± 5.32 vs 5.51 ± 3.40, P < 0.05). Expression of FOXP3 and CTLA-4 was also significantly higher in HCC patients ( P < 0.05) compared to CH group. Conclusions: We provide evidence of an increased population of Treg not only in the PB but also in tumor microenvironment of HCC patients, suggesting association of enhanced Treg activity with poor immune responses to tumor antigens. These findings may in future play a significant role in designing immunotherapeutic approaches in HCC.

Effect of all-trans retinoic acid [ATRA] on viability, proliferation, activation and lineage-specific transcription factors of CD4+ T cells

All-trans retinoic acid [ATRA], as an active metabolite of vitamin A, has been shown to affect immune cells. This study was performed to evaluate the effect of ATRA on viability, proliferation, activation and lineage-specific transcription factors of CD4+ T cells. CD4+ T cells were separated from heparinized blood of healthy donors and were cultured in conditions, some with, some without ATRA. Viability was assessed by PI flowcytometry and proliferation was measured by MTT assay. CD69 expression was determined by flowcytometry as a measure of cell activation. Lineage-specific transcription factors [FOXP3, RORgammat and T-bet] were examined by intracellular staining and flowcytometry. High doses of ATRA [0.1-1 mM] caused extensive cell death in both PBMCs and CD4+ T cells. Doses of ATRA equal to or lower than 10 microM did not adversely affect cell viability and proliferation in comparison to culture medium without ATRA. Doses of ATRA between 10 microM and InM significantly increased cell activation when compared to culture medium without ATRA. ATRA could increase FOXP3+ and also FOXP3+RORgammat+ T cells while it decreased RORgammat+ and T-bet+ T cells. This study showed that doses of ATRA up to 10 microM are safe when using with CD4+ T cells in terms of cell viability, proliferation and activation. We could also show that ATRA diverts the human immune response in neutral conditions [without adding polarizing cytokines] by increasing FOXP3+ cells and decreasing RORgammat+ cells. ATRA could be regarded as a potential therapy in inflammatory conditions and autoimmunities

Forkhead box O-class 1 and Forkhead box G1 as Prognostic Markers for Bladder Cancer

Forkhead box O-class 1 (FOXO1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. Its functions are modulated by forkhead box G1 (FOXG1), which acts as a transcriptional repressor with oncogenic potential. Real-time PCR and immunohistochemical staining were performed in 174 primary bladder cancer specimens and 21 normal bladder mucosae to evaluate these genes. FOXO1 and FOXG1 mRNA expression in cancer tissues were higher than in normal mucosae (each P<0.001). FOXO1 mRNA levels were significantly higher in samples of non-progressed patients (P<0.001), but FOXG1 were enhanced in those of progressed patients (P=0.019). On univariate analysis, FOXO1 mRNA expression was significantly associated with grade, stage, recurrence, progression and survival (each P<0.05). On multivariate analysis, increased FOXO1 mRNA expression was associated with both reduced disease progression (odds ratio [OR], 0.367; 95% confidence interval [CI], 0.163-0.826, P=0.015) and enhanced disease-free survival (OR, 3.262; 95% CI, 1.361-7.820, P=0.008). At a median follow-up of 33 months (range 2 to 156), the patients with a high FOXO1 mRNA expression had a significantly prolonged survival (P=0.001). Immunohistochemical findings of FOXO1 were generally concordant with mRNA expression levels. In conclusion, FOXO1 may be a promising marker for predicting progression in human bladder cancers.

Forkhead box O-class 1 and Forkhead box G1 as Prognostic Markers for Bladder Cancer

Forkhead box O-class 1 (FOXO1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. Its functions are modulated by forkhead box G1 (FOXG1), which acts as a transcriptional repressor with oncogenic potential. Real-time PCR and immunohistochemical staining were performed in 174 primary bladder cancer specimens and 21 normal bladder mucosae to evaluate these genes. FOXO1 and FOXG1 mRNA expression in cancer tissues were higher than in normal mucosae (each P<0.001). FOXO1 mRNA levels were significantly higher in samples of non-progressed patients (P<0.001), but FOXG1 were enhanced in those of progressed patients (P=0.019). On univariate analysis, FOXO1 mRNA expression was significantly associated with grade, stage, recurrence, progression and survival (each P<0.05). On multivariate analysis, increased FOXO1 mRNA expression was associated with both reduced disease progression (odds ratio [OR], 0.367; 95% confidence interval [CI], 0.163-0.826, P=0.015) and enhanced disease-free survival (OR, 3.262; 95% CI, 1.361-7.820, P=0.008). At a median follow-up of 33 months (range 2 to 156), the patients with a high FOXO1 mRNA expression had a significantly prolonged survival (P=0.001). Immunohistochemical findings of FOXO1 were generally concordant with mRNA expression levels. In conclusion, FOXO1 may be a promising marker for predicting progression in human bladder cancers.