High Foxp3 Expression Research Articles

Th-17 Alloimmune Responses in Renal Allograft Biopsies From Recipients of Kidney Transplants Using Extended Criteria Donors During Acute T Cell–Mediated Rejection

Although renal transplantation using expanded criteria donors has become a common practice, immune responses related to immunosenescence in those kidney allografts have not been studied yet in humans. We performed a retrospective molecular analysis of the T cell immune response in 43 kidney biopsies from patients with acute T cell-mediated rejection including 25 from recipients engrafted with a kidney from expanded criteria donor and 18 from recipients grafted with optimal kidney allograft. The clinical, transplant and acute T cell-mediated rejection characteristics of both groups were similar at baseline. The expression of RORγt, Il-17 and T-bet mRNA was significantly higher in the elderly than in the optimal group (p = 0.02, p = 0.036, and p = 0.01, respectively). Foxp3 mRNA levels were significantly higher in elderly patients experiencing successful acute T cell-mediated rejection reversal (p = 0.03). The presence of IL-17 mRNA was strongly associated with nonsuccessful reversal in elderly patients (p = 0.008). Patients with mRNA IL17 expression detection and low mRNA Foxp3 expression experienced significantly more treatment failure (87.5%) than patients with no mRNA IL17 expression and/or high mRNA Foxp3 expression (26.7%; p = 0.017). Our study suggests that the Th17 pathway is involved in pathogenesis and prognosis of acute T cell-mediated rejection in recipients of expanded criteria allograft.

Roles of Tumor-infiltrating Lymphocytes in Non-small Cell Lung Cancer Recurrence and Metastasis:A Meta Analysis

To determine the prognostic value of tumor-infiltrating lymphocytes in the recurrence and metastasis of non-small cell lung cancer (NSCLC). A PubMed, EMBASE, Cochrane Library databases, NIH databases, and China Biology Medicine disc, China National Knowledge Infrastructure, Chinese Science and Technology Periodical literature search strategy was designed. Studies on the prognostic values of intratumoural CD3⁺,CD4⁺,CD8⁺, and FoxP3+lymphocytes for NSLCL were retrieved. RevMan 5.1 was applied for Meta analysis. Totally 8 studies entered the final analysis. In pooled analysis of 1197 patients,the high expressions of CD3⁺ and CD8⁺ cells were correlated with the increase of overall survival (OS) (OR=0.52,95% CI=0.40- 0.68, P<0.0001; OR=0.52,95% CI=0.34-0.79,P=0.002), and the high expression of CD8⁺ was significantly correlated with the increase of disease-free survival (DFS) (OR=0.68,95% CI=0.51-0.91,P=0.01). The CD4⁺ cell expression level was not significantly correlated with OS or DFS (P=0.14, P=0.73). The high expression of FoxP3⁺ cells did not favor the DFS(OR=1.78,95% CI=1.36-2.31; P<0.0001). The expression levels of CD3⁺, CD8⁺ and FoxP3⁺ in NSCLC microenviroment are related with the prognosis of NSCLC, while the role of CD4⁺ in the development of NSCLC warrants further investigations.

Abstract 1284: Analysis of the immune suppression mechanism in extra-hepatic cholangiocarcinoma

Abstract Introduction Extra-hepatic cholangiocarcinoma (EHCC) has not been improved with the five-year survival rate for more than 10 years, which is a refractory cancer of poor prognosis. Development of novel therapeutic agents has been desired, because the standard chemotherapy has not been established yet. Anti-tumor immunotherapy is expected for less burden on the patient in recent years. However, it is limited by multiple tolerogenic mechanisms related to the tumor, the host immune system, and the tumor microenvironment. Therefore satisfactory results are not obtained in the clinical trials for several vaccinations. In this experiment, we focused on negative co-signaling molecules, especially immune checkpoints. The purpose of the present study was to investigate the relationship between the expression of immune checkpoints and clinico-pathological findings, including prognosis in EHCC patients. Patients and specimens We enrolled 81 patients (56 men and 25 women; age range: 36-84 years) with EHCC who underwent curative resection with lymphadenectomy at Gunma University Hospital and Saiseikai Maebashi Hospital (Maebashi, Japan) between 2000 and 2011. Patients who had undergone palliative resection, preoperative chemotherapy and radiation therapy were excluded from this study. Furthermore, none of the patients enrolled in this study had synchronous or metachronous cancer in other organs. Results Immunohistochemical staining of immune checkpoints in primary EHCC tissue was performed. Patients with high B7-H4 expression had a significantly poorer prognosis than those with low B7-H4 expression. However, it was not seen in B7-H3 expression in this experiment. Furthermore, in analysis for the expression of CD8, IFN-γ and FOXP3, patients with high FOXP3 expression had a significantly poorer prognosis than those with low FOXP3 expression. Conclusion The B7-H4 is a negative co-stimulatory molecule and a novel prognostic marker related to the T-cell mediated immune response. It's pathway may be a molecular target for controlling tumor progression in patients with EHCC. Citation Format: Satoshi Wada, Mariko Tsukagoshi, Akira Watanabe, Norio Kubo, Kenichiro Araki, Hideki Suzuki, Hiroyuki Kuwano. Analysis of the immune suppression mechanism in extra-hepatic cholangiocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1284. doi:10.1158/1538-7445.AM2015-1284

Regulatory T cells in ankylosing spondylitis and the response after adalimumab treatment

ObjectivesThe aim of this study was to investigate the role of regulatory T cells (Tregs) in ankylosing spondylitis (AS). MethodsWe included 69 AS patients (15 of them received anti-tumor necrosis factor-apha agent-adalimumab) in the study and used a questionnaire to record the demographic data, disease activity index, functional index, human leukocyte antigen-B27, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). Thirty healthy subjects were used as controls. The peripheral blood mononuclear cells (PMBCs) were stained with anti-CD4, anti-CD25 and anti-Forkhead/winged helix transcription factor P3 (anti-FoxP3) antibodies and flow-cytometry was used to determine cell populations. ResultsThe percentages of Tregs in PMBCs were significantly higher in AS patients than in healthy controls. In AS patients who had poor disease functional index with higher levels of ESR and CRP were positively and significantly correlated with Tregs percentages in PMBCs. After adalimumab treatment in 15 patients, the percentages of Tregs, the ESR/CRP levels and the Bath Ankylosing Spondylitis Disease Activity Index/Bath Ankylosing Spondylitis Functional Index were significantly and gradually decreased over time. ConclusionsThe high expression of FoxP3 and CD25 on CD4+ T cells in PBMCs in AS patients was noted, and could be reversed by adalimumab therapy. These findings suggest that Tregs may play a role in modulating the inflammatory process in AS. Whether Tregs can be taken as a predictor for disease activity or treatment outcome is unclear and requires further study.

Analysis of cytokine production by peanut-reactive T cells identifies residual Th2 effectors in highly allergic children who received peanut oral immunotherapy.

Only limited evidence is available regarding the cytokine repertoire of effector T cells associated with peanut allergy, and how these responses relate to IgE antibodies to peanut components. To interrogate T cell effector cytokine populations induced by Ara h 1 and Ara h 2 among peanut allergic (PA) children in the context of IgE and to evaluate their modulation during oral immunotherapy (OIT). Peanut-reactive effector T cells were analysed in conjunction with specific IgE profiles in PA children using intracellular staining and multiplex assay. Cytokine-expressing T cell subpopulations were visualized using SPICE. Ara h 2 dominated the antibody response to peanut as judged by prevalence and quantity among a cohort of children with IgE to peanut. High IgE (> 15 kU(A)/L) was almost exclusively associated with dual sensitization to Ara h 1 and Ara h 2 and was age independent. Among PA children, IL-4-biased responses to both major allergens were induced, regardless of whether IgE antibodies to Ara h 1 were present. Among subjects receiving OIT in whom high IgE was maintained, Th2 reactivity to peanut components persisted despite clinical desensitization and modulation of allergen-specific immune parameters including augmented specific IgG4 antibodies, Th1 skewing and enhanced IL-10. The complexity of cytokine-positive subpopulations within peanut-reactive IL-4(+) and IFN-γ(+) T cells was similar to that observed in those who received no OIT, but was modified with extended therapy. Nonetheless, high Foxp3 expression was a distinguishing feature of peanut-reactive IL-4(+) T cells irrespective of OIT, and a correlate of their ability to secrete type 2 cytokines. Although total numbers of peanut-reactive IL-4(+) and IFN-γ(+) T cells are modulated by OIT in highly allergic children, complex T cell populations with pathogenic potential persist in the presence of recognized immune markers of successful immunotherapy.

Association of intratumoral regulatory T-cell accumulation in patients with hepatocellular carcinoma (HCC) with poor survival: Effect of autologous dendritic cell immunotherapy on selective reduction of regulatory T cells and survival.

e14023 Background: The immune status of the tumor microenvironment influences tumor progression in HCC. Regulatory T cells (Tregs) inhibit effector cell responses, and play a pivotal role in effective cancer surveillance. This study examines the role on overall survival of intratumoral Tregs in patients with HCC, and evaluates the impact of autologous dendritic cell (DC) immunotherapy on Treg depletion, tumor progression, and overall survival in an orthotopic murine HCC model. Methods: The expression of Foxp3 and CD4 in 63 paired tumor and non-neoplastic liver resection tissue specimens of HCC patients was evaluated using RT-PCR. Orthotopic murine HCC was established by implanting Hepa1-6 cells in the liver. The effect of DC immunotherapy loaded with Hepa1-6 cell lysate on tumor progression, survival, tumor infiltrating lymphocytes and cytokines was examined. Results: High intratumoral Foxp3 expression in HCC patients treated with resection was associated with poor overall survival. DC immunotherapy in HCC bearing mice resulted in reduced Foxp3+CD4+ regulatory T cell accumulation and TGF-b production within the tumor microenvironment. Moreover, DC immunotherapy reversed murine HCC progression, as assessed by plasma a-fetoprotein levels and in vivo bioluminescence imaging, and increased survival to 90% by day 60, as compared to < 5% in untreated tumor-bearing mice. This anti-tumor response was accompanied by inhibition of STAT3 phosphorylation within tumor tissue Conclusions: High levels of intratumoral Tregs in patients undergoing hepatectomy is with associated poor overall survival. Tumor cell lysate-loaded DC immunotherapy results in tumor regression and increased survival in a clinically relevant orthotopic murine HCC model. The inhibitory effect of DC immunotherapy on the accumulation of Foxp3+CD4+ regulatory T cells within the tumor microenvironment and on the production of TGF-b suggests that effective DC immunotherapy can alter the immunosuppressive tumor microenvironment, thus resulting in tumor regression.

Human in vitro induced T regulatory cells and memory T cells share common demethylation of specific FOXP3 promoter region.

BackgroundThe FOXP3 gene is the master regulator for T regulatory cells and is under tight DNA methylation control at the Treg specific demethylated region (TSDR) in its first intron. This said, methylation of its promoter region, the significance of which is unknown, has also been associated with various immune-related disease states such as asthma, food allergy, auto-immunity and cancer. Here, we used induced T regulatory cells (iTreg) as a target cell population to identify candidate hypomethylated CpG sites in the FOXP3 gene promoter to design a DNA methylation quantitative assay for this region.FindingsThree CpG sites at the promoter region showed clear demethylation pattern associated with high FOXP3 expression after activation in presence of TGFβ and were selected as primary targets to design methylation-dependent RT-PCR primers and probes. We then examined the methylation of this ‘inducible-promoter-demethylated-region’ (IPDR) in various FOXP3+ T cell subsets. Both naïve and memory thymic-derived Treg cells were found to be fully demethylated at both the IPDR and TSDR. Interestingly, in addition to iTregs, both CD25− and CD25lo conventional memory CD4+CD45RA− T cells displayed a high fraction of IPDR demethylated cells in absence of TSDR demethylation.ConclusionThis implies that the fraction of memory T cells should be taken in account when interpreting FOXP3 promoter methylation results from clinical studies. This approach, which is available for testing in clinical samples could have diagnostic and prognostic value in patients with immune or auto-inflammatory diseases.

Tumor Microenvironmental Features and Outcome in Post-Transplant Lymphoproliferative Disorder

Background: PTLD comprises a diverse spectrum of hematological conditions, ranging from early lesions, characterized by reactive-like proliferations, to monomorphic lesions, resembling overt lymphoma. Â In most cases, the lesions are believed to arise as the result of reduced immune surveillance secondary to the use of immunosuppressive drugs post-transplant. This view is supported by the observation that PTLDs, particularly those characterized by early or polymorphic lesions, may regress spontaneously upon reduction of the immunosuppressive treatment. Studies in sporadic lymphomas have identified distinct microenvironmental characteristics, predictive of the clinical behavior, but data is scarce in the immunocompromised setting. Therefore, the aim of this study was to investigate the tumor microenvironment in a population-based cohort of PTLD.Methods: We identified 108 PTLD patients diagnosed in the period 1994-2011. Of these, 62 cases had adequate tissue for tissue microarray construction. All biopsies were reviewed and classified according to the WHO 2008 criteria. Immunohistochemically stained sections were digitally quantified using Tissuemorph (Visiopharm Integrator System 4.0.3.0, Visiopharm, Denmark). Determination of optimal cut-off values of the area fraction (AF) was established by a ROC-curve. ROC analyses were performed in every disease entity and used for endpoint analyses.Results: Themedian age was 45 yrs (range 2-77 yrs) with a M/F ratio of 3:1. The majority presented in stage I-II (61%), and B symptoms and extranodal disease were common features (40% and 42%, respectively). Most tissue samples were EBV-positive (85%). The EBV latency pattern was predominantly latency II (41%) or III (43%). The AF of galectin-1 (gal-1) positive cells was clearly correlated to latency type (p=0.0001), whereas FOXP3 and programmed death-1 (PD-1) did not show such correlation (fig 1).Overall survival (OS) was significantly higher in cases with high levels of PD-1 expression, with 5-yrs OS of 39% (23-55%) and 69% (47-83%) for low and high levels, respectively (p=0.01). Expression levels of FOXP3 and gal-1 had no impact on OS in the total cohort (fig 1).In the monomorphic setting, a low AF of FOXP3 positive cells was associated with an increased risk of progression (OR 6.1; 95% CI: 1.4-26.0). This did not, however, translate into an inferior OS (p=0.163).To specifically characterize the tumor microenvironmental features of DLBCL-type PTLD with respect to cell of origin (COO), 30 evaluable cases were analyzed according to the Hans classifier; 10 (33%) were germinal center (GC) type and 20 (67%) of non-GC type. Cases of non-GC subtype had a significantly shorter time to PTLD of 1.16 yrs (CI: 0.64- 2.11), than those of GC-subtype (3.66 yrs; CI: 1.64- 8.16) (p=0.023) and a lower AF of gal-1 positive cells (p=0.042). The 5-yrs OS was 58% (32-77%) and 0% for non-GC versus GC-tumors, respectively (p=0.075). High levels of FOXP3 expression were associated with superior OS in the non-GC sub-group (p=0.04); gal-1 and PD-1 had no influence in the COO setting.Conclusion: The present study is one of the few attempts to describe tumor microenvironmental features in PTLD and relate them to outcome. In a population-based PTLD cohort, we found that specific immune cell subsets were variably expressed in different PTLD subtypes, and that high expression of PD-1 (whole cohort) and FOXP3 (non-GC DLBCL only) correlated with significantly better outcome. ÂN(%) pts in TMAOrgan tx(%)Kidney Heart Lung47(75.8) 9(14.5) 6(9.7)Type PTLD(%)Early/polymorphic lesions Monomorphic PTLD Diffuse large B-cell lymphomaPeripheral T-cel lymphomal, NOS*T-Anaplastic large cell lymphomaBurkitt lymphomaHodgkin lymphoma-type PTLD Marginal zone lymphoma18(29.0) 38(61.2) 32(51.6) 1(1.6) 3(4.8) 2(3.2) 4(6.5) 2(3.2) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mycobacterium tuberculosis modulates the gene interactions to activate the HIV replication and faster disease progression in a co-infected host.

Understanding of the chronic immune activation, breakdown of immune defense and synergistic effect between HIV and Mycobacterium tuberculosis (Mtb) may provide essential information regarding key factors involved in the pathogenesis of HIV disease. In this study, we aimed to highlight a few of the immunological events that may influence and accelerate the progression of HIV disease in the presence of co-infecting Mtb. A cross-sectional study was performed on cohorts, including anti-tubercular therapy (ATT) naïve active pulmonary tuberculosis (PTB) patients, antiretroviral therapy (ART) naïve HIV-1 infected individuals at different stages of disease, ATT and ART naïve HIV-PTB co-infected individuals and healthy controls. A significantly higher T-regulatory cell (Treg) frequency coupled with the high FoxP3 expression in the CD4 T-cells indicated an immunosuppressive environment in the advance stage of HIV-1 infection. This is further substantiated by high HO-1 expression favoring TB co-infection. Functionally, this change in Treg frequency in HIV-1 infected individuals correlated well with suppression of T-cell proliferation. Mtb infection seems to facilitate the expansion of the Treg pool along with increased expression of FoxP3, specifically the variant-1, as evident from the data in HIV-1 co-infected as well as in patients with only PTB. A significantly lower expression of HO-1 in co-infected individuals compared to patients with only HIV-infection having comparable CD4 count correlated well with increased expression of CCR5 and CxCR4 as well as NF-κB and inflammatory cytokines IL-6 and TNF-α, which collectively may contribute to enhanced viral replication and increased cell death, hence faster disease progression in co-infected individuals.

Combination of ATG and Rapamycin Prolongs the Persistence of Adoptively-Transferred, Ex Vivo-Expanded Third Party Treg in Cynomolgus Monkeys.

Background: It has been shown that ex vivo-expanded regulatory T cells (Treg) can promote transplant tolerance in small animal models as well as in phase I clinical trials of hematopoietic stem cell transplantation. The issue has been raised of establishing Treg banks for adoptive cell therapy of organ transplant rejection and autoimmune disease. However, little is known about the persistence, stability and fate of infused Treg, especially third-party allogeneic Treg, and the influence of T cell-depleting and other immunosuppressive agents on their survival and therapeutic efficacy. Methods and Results: In this study, we flow-sorted CD4+CD25+CD127- Treg from normal juvenile cynomolgus monkey PBMC and expanded these cells using a protocol with artificial antigen-presenting cells. Three rounds of stimulation expanded the cells over 1000-fold and consistently generated over 108 Treg from 30 ml peripheral blood, while maintaining expression of Foxp3, CD25, CTLA-4, Helios; potent capacity to suppress T cell proliferation in vitro; demethylation status at the Treg-Specific Demethylation Region (TSDR), and down-regulated expression of CD45RA, CCR7, CD62L and upregulation of CXCR3 suggesting that these cells are likely to migrate to inflamed sites immediately after infusion in vivo. More importantly, when CFSE-labeled autologous (auto) and VPD450-labeled allogeneic (allo) ex vivo expanded Tregs were transferred simultaneously into monkeys, the number of auto-Treg was up to 100 per ml peripheral blood for at least 6 days (ie. for the duration of the experiment) in both control and ATG/rapamycin-treated (IS) monkeys. In contrast, by day 3 after their infusion, allo-Treg could not be detected in control monkeys, while they were maintained up to 100 cells per ml peripheral blood for at least 15 days in IS monkeys. These Tregs maintain high expression of Foxp3, CD25, and CXCR3 expression, and low expression of CD127, CCR7, and CD62L. Conclusion: The finding that allo-Treg can survive in IS-treated monkeys and maintain Treg phenotype supports the concept and potential utility of Treg banks for clinical application in transplantation and autoimmune disease and provides valuable information required to take allo-Treg therapy towards phase II/III clinical trials for efficacy assessment.

Effect of HIF1α on Foxp3 expression in CD4+ CD25- T lymphocytes.

The aim of the present study was to investigate the effect of HIF1α on Foxp3 expression in CD4(+) CD25(-) T lymphocytes. CD4(+) CD25(-) T lymphocytes were sorted from PBMC using a CD4(+) CD25(+) regulatory T cell isolation kit. Lentivirus containing lentiviral vector that overexpressed HIF1α (HIF-lenti) and those containing empty expression vector (control-lenti) were produced. Meanwhile, lentivirus that contained lentiviral vector that suppressed HIF1α expression (siHIF-lenti) and those containing control vector (sicontrol-lenti) were also generated. The sorted CD4(+) CD25(-) T lymphocytes were infected with HIF-lenti, control-lenti, siHIF-lenti, and sicontrol-lenti, respectively. Approximately 72 hr after transduction, real-time PCR and Western blot were carried out to analyze the RNA and protein expression level of HIF1α and Foxp3. CD4(+) CD25(-) T lymphocytes cultured under 21% O2 , 5% CO2 (normoxia) and 1% O2 , 5% CO2 (hypoxia) were used as control. Our results showed that overexpression of HIF1α increased both mRNA and protein expression of Foxp3 and, meanwhile, suppression of HIF1α expression by RNAi could reverse high Foxp3 expression in CD4(+) CD25(-) T lymphocytes caused by hypoxic culture. These results suggested that hypoxia could stimulate Foxp3 expression by increasing HIF1α expression in CD4(+) T lymphocytes which may promote CD4(+) T lymphocytes to convert to Treg.

Expression of costimulatory molecules B7-H1, B7-H4 and Foxp3+ Tregs in gastric cancer and its clinical significance

Immune escape plays an important role in tumor progression. In the present study, the expression of B7-H1, B7-H4 and Foxp3 involved in immune escape in gastric carcinoma was investigated and the corresponding clinical significance was evaluated. Immunohistochemistry was used to detect the expression of B7-H1, B7-H4 and Foxp3 in 100 gastric cancer specimens, and 30 paracarcinoma tissues were used as the control. Both B7-H1 and B7-H4 showed high expression levels in gastric cancer tissues (65.0 and 71.0 %, respectively), and the expressions of B7-H1 and B7-H4 were positively correlated with the depth of tumor invasion, lymph node metastasis and American Joint Committee on Cancer (AJCC) stage (P < 0.05). The number of Foxp3(+) Tregs was much higher in gastric cancer tissues than control tissues, which was positively correlated with lymph node metastasis (P < 0.05). Similarly, a positive correlation between B7-H1 or B7-H4 expression and the number of Foxp3(+) Tregs was observed. The median overall survival rate of patients with high expression of B7-H1, B7-H4 and Foxp3 was significantly poorer than that of patients with low expression of these proteins (P < 0.05). Cox regression multivariate analysis confirmed that lymph node metastasis, AJCC stage, and B7-H1 and Foxp3 overexpression were independent prognostic factors. B7-H1, B7-H4 and Foxp3 were overexpressed in gastric cancer tissues. B7-H1 and Foxp3 are negative prognostic factors for patients with gastric cancer.

Comparative Characterization of Stroma Cells and Ductal Epithelium in Chronic Pancreatitis and Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive stroma being also present in chronic pancreatitis (CP). Using immunohistochemistry, the stroma of CP and PDAC was comprehensively analyzed and correlated with epithelial/carcinoma-related alterations and clinicopathological patient characteristics. While there were no significant differences between CP and PDAC regarding the distribution of CD3+ T cells and α-SMA+ fibroblasts, proportions of CD4+ and CD8+ T cells were significantly lower and numbers of CD25+(CD4+) and FoxP3+(CD4+) regulatory T cells were greater in PDAC compared with CP. Macrophages were more prevalent in CP, but localized more closely to carcinoma cells in PDAC, as were γδ-T cells. Duct-related FoxP3 and L1CAM expression increased from CP to PDAC, while vimentin expression was similarly abundant in both diseases. Moreover, stromal and epithelial compartments of well-differentiated tumors and CPs shared considerable similarities, while moderately and poorly differentiated tumors significantly differed from CP tissues. Analysis of 27 parameters within each pancreatic disease revealed a significant correlation of i) CD4+ and FoxP3+CD4+ T cells with FoxP3 expression in PDAC cells, ii) α-SMA+ fibroblasts with L1CAM expression and proliferation in PDAC cells, iii) CD3 and CD8 expression with γδ-TCR expression in both pancreatic diseases and iv) CD68+ and CD163+ macrophages with vimentin expression in PDAC cells. High expression of FoxP3, vimentin and L1CAM in PDAC cells as well as a tumor-related localization of macrophages each tended to correlate with higher tumor grade. Multivariate survival analysis revealed a younger age at time of surgery as a positive prognostic marker for PDAC patients with the most frequently operated disease stage T3N1M0. Overall this study identified several interrelationships between stroma and epithelial/carcinoma cells in PDACs but also in CP, which in light of previous experimental data strongly support the view that the inflammatory stroma contributes to malignancy-associated alterations already in precursor cells during CP.

Immune alterations in lung cancer - the new therapeutic approach

Lung cancer is the main cause of cancer death worldwide. An advanced stage of the disease at the time of diagnosis, observed in the majority of cases, does not allow for introduction of radical treatment or makes the treatment ineffective. Lung cancer as a solid tumour with a very low antigenicity escapes immune surveillance, and cytotoxic cells attack. Cytotoxic lymphocytes play a key role in anticancer defence, but the population of these cells individually differs. Many suppressor and regulatory mechanisms inhibit the recognition of tumour antigens by dendritic and cytotoxic cell activation. The population of regulatory T cells (T regs) plays a crucial role in this inhibition of immune response. Their function depends on the expression of transcription factor Foxp3 and the presence of CTLA-4 molecules. The increased proportion of T regs and high expression of Foxp3 and CTLA-4 on lung cancer cells and tumour infiltrating lymphocytes were observed. Other components of immune response inhibition and tumour promotion are: Th17 cell population, M2 macrophage polarisation, the presence of myeloid derived suppressor cells (MDSCs) and a significantly elevated concentration of cytokines: TGF-b and IL-10 in the tumour microenvironment. The recognition of these mechanisms may have important therapeutic implications. Several types of agents which are capable of modulating the immune response have recently been used in many clinical trials conducted in lung cancer patients, some of them showing efficacy. Lung cancer immunotherapy has two main directions: the first goal is to improve the cytotoxic effect (for example by inhibition of CTLA-4, stimulation of dendritic cell function, inhibition of lymphocyte apoptosis), and the second way is the production of anti-cancer vaccines using known cancer antigens: MAGE A3, MUC1, EGF and TGF-b. Immunotherapy in lung cancer treatment has a character of personalised therapy - there is a need to specify the patient's immune status prior to treatment. The analysis of immune cells and mediators in the peripheral blood allows this, but the more valuable method seems to be bronchoalveolar lavage (BAL) examination with careful assessment of the tumour microenvironment.

Expression of CXC chemokine receptor-4 and forkhead box 3 in neuroblastoma cells and response to chemotherapy.

Current evidence indicates that the abnormal expression of chemokines or their receptors, such as CXC chemokine receptor-4 (CXCR4), is positively correlated with the development, progression and metastasis of tumor cells. However, the role of CXCR4 in neuroblastoma and its response to chemotherapy remain largely unclear. In addition, forkhead box 3 (Foxp3), a transcription factor associated with T cell tolerance, is expressed in tumor cells and plays a role in the immune evasion of cancers. The present study aimed to examine the expression of CXCR4 and Foxp3 in the LAN-5 and SK-N-SH neuroblastoma cell lines. The effects of chemotherapy drugs, cyclophosphamide (CTX) and pirarubicin (THP), on the expression of these two genes were also investigated. Our findings indicated that CXCR4 and Foxp3 were highly expressed in LAN-5 and SK-N-SH cells. Following treatment with CTX and THP, the protein expression of CXCR4 in LAN-5 and SK-N-SH cells was significantly decreased (P<0.05). The expression of Foxp3 in LAN-5 cells was also significantly downregulated by CTX and THP treatment (P<0.05). Therefore, the high expression of CXCR4 and Foxp3 in LAN-5 and SK-N-SH cells and their subsequent downregulation following administration of the chemotherapy agents suggests that the chemokine receptors, CXCR4 and Foxp3, may be involved in the metastasis and tumor evasion of neuroblastoma. Further studies should investigate the expression of CXCR4 and Foxp3 in patient samples.

Targeting PKC in Human T Cells Using Sotrastaurin (AEB071) Preserves Regulatory T Cells and Prevents IL-17 Production

Regulatory T-cells (Treg) are crucial for immune homeostasis and prevention of immune pathology. Yet, Treg may lose Foxp3 and start secreting IL-17, dependent on environmental cues. Our previous data revealed that Treg from severe psoriasis patients are particularly prone to such conversion. The question of how to maintain Treg stability in the context of inflammation awaits immediate resolution. The pan-protein kinase C (PKC) inhibitor sotrastaurin has shown efficacy in clinical trials of psoriasis. Here, we show that sotrastaurin inhibited effector T-cell responses, whereas the regulatory response was enhanced. Sotrastaurin prevented TCR/CD28-induced T-cell activation and pro-inflammatory cytokine production, but preserved a stable Treg phenotype as evidenced by maintenance of suppressive capacity, high Foxp3 and CD25 expression, and lack of IL-17A and IFNγ production. Moreover, in both circulating and dermal psoriatic Treg, prone to rapid induction of IL-17, sotrastaurin enhanced Foxp3 expression and prevented IL-17A and IFNγ production even when stimulated in the presence of the helper T 17-enhancing cytokines IL-1β or IL-23. Thus, pharmacological inhibition of PKC may serve as a powerful tool to concurrently inhibit effector T cells and to facilitate Treg, thereby showing therapeutic potential for the treatment of psoriasis.

The role of tumour FoxP3 as prognostic marker in different subtypes of head and neck cancer

Expression of the forkhead transcription factor (FoxP3) – an established marker of regulatory T cells – has been found in other cell types as well, including tumour cells. Recent studies indicated that high tumour FoxP3 expression might be associated with a poor outcome of patients with several types of solid cancers. Here, we investigated the role of FoxP3 expressed by the tumour cells in the prognosis of larynx and oro-hypopharynx squamous cell carcinoma (LSCC and OHSCC) – two major subtypes of head and neck cancer. To this end, we analysed by immunohistochemistry the expression of tumour FoxP3 in tissues from 83 LSCC and 89 OHSCC patients in relation to overall survival. In multivariate analysis we found that high tumour FoxP3 expression significantly associated with poor survival in OHSCC but not in LSCC patients. In further studies, we combined the prognostic value of FoxP3 with selected markers of inflammation (cyclooxygenase-2; COX2) or with markers of enhanced tumour migration/invasion (AHNAK and CORTACTIN). Interestingly, we found that the combination of FoxP3 and AHNAK (in LSCC) or FoxP3 and CORTACTIN (in OHSCC) had significantly stronger prognostic values than either marker analysed individually. Combination of FoxP3 and COX2 enhanced the prognostic accuracy only in OHSCC. Thus, our study identifies novel individual and combination markers that might have enhanced and distinct prognostic relevance in different subtypes of head and neck cancer.

Expression and clinical significance of costimulatory molecules B7-H1, B7-H4 and forkhead box P3+ regulatory T cells in gastric carcinoma

Objective To investigate the expression of B7-H1,B7-H4 and forkhead box P3 (Foxp3) in gastric carcinoma and its clinical significance.Methods The expression of B7-H1,B7-H4 and Foxp3 was detected by using IHC in 100 gastric carcinomas,and the relationship between the expression of B7-H1,B7-H4 and Foxp3 with clinicopathological features was statistically analyzed.Results Both B7-H1 and B7-H4 showed high expression in gastric carcinoma tissues (65.0％ and 71.0％).The expression of B7-H1 and B7-H4 proteins was positively correlated to the depth of tumor invasion,lymph node metastasis and TNM stage.The positive expression rate of B7-H1 and B7-H4 in the patient with less than 5-year survival was obviously higher than that in the patients with more than 5-year survival (P ＜ 0.05).High expression of Foxp3 + regulatory T cells (Treg) cells in gastric cancer tissues was correlated to lymph node metastasis (P ＜ 0.05).Statistical analysis also showed that the expression of B7-H1 and B7-H4 protein was positively correlated to the number of Foxp3 + Treg cells (r =0.437,P ＜ 0.05 ; r =0.456,P ＜0.05).Conclusion B7-H1,B7-H4 and Foxp3 + Treg cells were up-regulated in gastric cancer tissues,and may be correlated to gastric carcinoma metastasis and prognosis. Key words: Gastric cancer; Costimulatory molecule; Forkhead box P3

High FoxP3 expression in tumour cells predicts better survival in gastric cancer and its role in tumour microenvironment

Background:Forkhead Box P3 (FoxP3) is thought to be a key transcription factor in regulatory T cells (Tregs), and recent data indicate that it is expressed in several tumour cells. However, its precise roles in gastric cancer (GC) and the underlying mechanisms regulating the interaction between GC cells and lymphocytes remain unclear.Methods:FoxP3 expression was examined in tumour cells and Tregs in 150 cases of gastric precancer and cancer, and their prognostic significances were evaluated, respectively, using a tissue microarray containing 135 GC patient samples with a mean 102-month follow-up. FoxP3 involvement in the tumour cells–lymphocytes interaction and its gene function were further investigated.Results:strong cytoplasmic staining of FoxP3 was observed in GC cells. FoxP3 protein expression in tumour cells predicts a good prognosis, whereas high-density Treg predicts a poor prognosis. Moreover, FoxP3 expression in GC cells increased after coculture with peripheral blood mononuclear cells through coculture systems. Upregulation of FoxP3 inhibited tumour growth in tumour-bearing nude mice.Conclusions:High FoxP3 expression in tumour cells predicts better survival in GC, possibility in relation to interaction between tumour cells and lymphocytes in microenvironment. Interfering with FoxP3 expression may open a new therapeutic strategy against tumour progression.

High expression of FOXP3 in primary melanoma is associated with tumour progression

The antitumour immune response plays an important role in the prognosis of melanoma. High numbers of circulating regulatory T cells have been associated with rapid disease progression. To assess the influence of forkhead box protein (FOXP)3, CD1a and langerin expression on the prognosis of primary melanoma. We analysed 185 primary melanomas by immunohistochemical staining for expression of the regulatory T-cell marker FOXP3 and the dendritic cell markers langerin and CD1a, and correlated marker expression with clinical outcome. Disease-free survival and overall survival were significantly longer in patients expressing low levels of FOXP3 in the primary melanoma, whereas they were associated with high expression of CD1a. The negative prognostic value of FOXP3 expression was independent of the Breslow tumour thickness. Langerin expression did not correlate with the clinical outcome. High expression of FOXP3 in the primary melanoma may be used as an additional independent prognostic marker for early tumour progression in patients with melanoma.