Foxp3 Gene Expression Levels Research Articles

Focusing on tumor and it’s microenvironmental immune members for head and neck cancer patients

ObjectiveImmune-related gene expression levels in the tumor microenvironment (TM) of head and neck squamous cell carcinoma (HNSCC) patients was compared. Materials and methodsThe CD163, CD274, CD86, FUT4, FOXP3, and ITGAX levels of HNSCC patients in their tumor tissues (n =76) and surrounding tissues adjacent to the tumor (n =76) were determined using quantitative real-time PCR (qRT-PCR). Changes in these genes were also evaluated by associating with demographical data of the patients. ResultsCD163, CD274, FUT4, and FOXP3 gene expression levels were significantly higher in tumor tissue than in surrounding tissue. FUT4 fold change was statistically higher in patients with lymph node involvement. CD86 expression was statistically lower in smokers of 50 boxes per year or more. CD163, CD274, and FUT4 expressions were increased in response to the presence of extranodal extension (ENE). ConclusionsThese preliminary results demonstrate the alterations in expression levels of immunologic markers are associated with the clinical presentations of HNSCC. Availability of data and materialsThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Improved Function and Maturation of Dendritic Cells Stimulated by Recombinant pp65 Protein: An in-vitro Design.

Stimulated dendritic cells (DCs) have been shown to be effective in the induction of specific immune cells. Also, the CMV pp65 plays an important role in CMV life cycle and immune recognition. To assess the effect of CMV pp65 on the maturity and function of dendritic cells. Splenic DCs were treated with non-cytotoxic concentrations of the pp65 and analyzed for MHC II, CD86, and CD40 expression by flow cytometry. Then, ROR-γ, GATA3, T-bet, and FOXP3 gene expression levels were evaluated in T cells co-cultured with DCs using Real time-PCR. Finally, the effects of pp65 on allogenic T-cell responses in mixed lymphocyte culture (MLR), and the release of cytokines were investigated by ELISA and flow cytometry. The phagocytosis rate was significantly lower in the pp65-treated DCs than the non-stimulated DCs. There were significant differences in the raised level of CD40, CD86, and CCR7 in DCs as maturation markers. Furthermore, ROR-γ, and T-bet overexpression in T cells of the pp65-treated group compared with the non-stimulated group was observed. Significant differences were observed in the levels of IL-2, IL-6, IL-17, IL-22, TNF-α, and IFN-γ in pp65-stimulated groups compared with the non-stimulated DCs. The pp65-treated DCs can induce differentiation and functional activity of the cellular immune system, including Th17, and Th1, but not other major T-cell subsets such as Tregs, and Th2 population.

Expression of Regulatory γδ T Cells in Patients with Acute Graft-Versus-Host Disease

Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the most effective therapy for hematologic malignancies. Acute graft-versus-host disease (aGVHD) is a major complication after allo-HSCT and leads to high transplant-related mortality. Regulatory γδ T cells (γδ Tregs), as a novel subset of γδ T cells with Foxp3 expression and immunosuppressive effect, are found to effectively alleviate aGVHD in mice model. However, the effect of γδ Tregs on the occurrence and development of aGVHD are not fully understood. The aim of this study is to investigate the expression levels and clinical significance of γδ Tregs in patients with aGVHD.Methods The immunophenotyping of γδ Tregs was analyzed in peripheral blood from 15 patients with aGVHD at the following two time points (at the onset of aGVHD and two weeks after the treatment), using flow cytometry. Twelve patients responded effectively to the treatment within two weeks, and 3 did not respond within two weeks. The expression levels of immunoregulatory-associated molecules (Foxp3, CD25, CTLA-4, GITR, TLR8, RORc, STAT-1 and STAT-3) were analyzed in peripheral blood, using real-time RT-PCR with SYBR GreenIstaining. Liquichip technology was used to analyze the concentration of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10, IL-17 and tumour necrosis factor-α (TNF-α) in cultural supernatant.Results The proportions of γδ T, Vδ1 T and Vδ2 T cells did not differ significantly between the untreated patients and those with effective treatment (P=0.308, P=0.117 and P=0.099). However, the proportions of Foxp3+γδ T, Foxp3+Vδ1 T and Foxp3+Vδ2 T cells were significantly increased after effective treatment (P=0.023, P=0.008 and P=0.028). The mRNA expression levels of Foxp3, RORc and TLR8 genes were significantly increased after effective treatment (P=0.060, P=0.001 and P=0.041). The expression levels of CD25, CTLA-4, GITR, STAT-1 and STAT-3 genes were similar between the untreated patients and those with effective treatment (P=0.95, P=0.421, P=0.605, P=0.186 and P=0.809). In addition, the concentration of IL-10 was decreased after effective treatment (P=0.005), and the concentration of IFN-γ, IL-4, IL-17 and TNF-α was similar between the two groups (P=0.662, P=0.314, P=0.152 and P=0.254). At the same time, we found that the proportion of Foxp3+γδ T cells and the expression level of Foxp3 gene both had a decreased tendency after ineffective treatment. The concentration of IL-10 had a increased tendency after ineffective treatment.Conclusions γδ Tregs might influence in the occurrence and development of aGVHD. DisclosuresLiu:Science and technology planning project of Guangdong Province (2014B020226004);: Research Funding; the project of health collaborative innovation of Guangzhou city (201400000003-1, 201400000003-4, 201508020254).: Research Funding; National Natural Science Foundation of China (81270647, 81300445, 81470349, 81400141, U1401221, 81401315).: Research Funding; Natural Science Foundation of Guangdong Province (2014A030310171, 2016A030310390) ;: Research Funding.

Abstract 4899: Tumor T-cell receptor (TCR) diversity elucidates the immune response to genetic alterations of muscle-invasive bladder cancer

Abstract Background: Muscle-invasive bladder cancer (MIBC), unlike the more favorable low-grade papillary form, has a dismal prognosis with a significant risk of recurrence after surgical resection. Previous whole-exome sequencing of MIBC tumors performed by our lab demonstrated that carriers of mutations in any of six DNA mismatch repair genes (ATM, ERCC2, FANCD2, PALB2, BRCA1 or BRCA2) not only had a high somatic mutation burden, but also had enhanced recurrence-free survival compared to non-carriers of these mutations. We hypothesize that a high somatic mutation burden may generate a higher number of tumor-specific antigens that induce clonal expansion of tumor-infiltrating T-lymphocytes (TILs) to reduce a risk of recurrence and improve the survival. To investigate this hypothesis, we have sequenced the TCR cDNAs of TILs. Methods: Tumor and adjacent normal tissue (verified by a pathologist) were collected from patients whose tumors had previously been whole-exome sequenced. Library preparation was performed using previously described methods. Next-generation sequencing of TCRs was conducted on the Illumina platform (San Diego, CA) and CDR3 sequences were identified using a published algorithm. The diversity index of the TCRα and TCRβ chains was calculated using Simpson's diversity index, an ecological formula for calculating biodiversity based on evenness and richness. CD4+, CD8+, and FOXP3 gene expression levels were also examined with TaqMan Gene Expression Assays (Thermo Fisher Scientific, Carlsbad, CA). Results: TCR sequencing of ten tumors (5 non-recurrent (NR) with at least two years follow-up, 5 recurrent) has been performed. The average TCRα DI of five recurrent patients is 97±56.5 (SEM) compared to 23± 12.7 for five NR patients. In addition, we found an average DI of 13.8±0.77 in tumors with a high somatic mutation burden (average number of mutations 221), while it was 71.4±36.8 for tumors with a low somatic mutation burden (avg mutations 31). In samples with a relatively high DI, the three most abundant TCR clones represent less than 15% of the total TCR population, while in samples with a low DI, the top three clones comprise 35-90% of the total population. Finally, the CD8+/FOXP3 ratios were 10.1±2.1(SEM) and 5.6±2.9 for tumors with a low DI and those with a high DI, respectively (low vs. high defined by the average). Conclusions: We have elicited a correlating pattern where MIBC tumors with a higher number of somatic mutations have a lower TCR DI, indicating an oligoclonal expansion. Furthermore, in patients with recurrence, the TCR DI is higher, suggesting a lack of anti-tumor immune responses and non-specific T-cell expansion. The higher CD8+/FOXP3 ratio observed in tumors with low DI suggests that the clonal expansion is of CD8+ cells in tumors, which have been shown to be associated with favorable prognosis. Citation Format: Noura Choudhury, Kai Lee Yap, Kazuma Kiyotani, Poh Yin Yew, Alexa Campanile, Tatjana Antic, Gary Steinberg, Jae-Hyun Park, Peter H. O'Donnell, Yusuke Nakamura. Tumor T-cell receptor (TCR) diversity elucidates the immune response to genetic alterations of muscle-invasive bladder cancer. [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 4899. doi:10.1158/1538-7445.AM2015-4899

Foxp3 gene polymorphisms and haplotypes associate with susceptibility of Graves' disease in Chinese Han population.

Foxp3 plays important roles in the pathogenesis of autoimmune diseases. To investigate the association between Foxp3 gene polymorphisms and the susceptibility to Graves' disease (GD) in Chinese Han population, four single nucleotide polymorphisms (SNPs) including -2383, -3279, -3499 in the promoter and IVS9+459 in the intron were genotyped. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 308 GD patients and 306 healthy controls. The relative expression level of Foxp3 gene was measured by qRT-PCR. The frequencies of AA/CA genotype of -3279 and CC genotype of IVS9+459 were significantly higher in GD patients than healthy controls. The AA/CA genotype of -3279 in female GD patients was more frequent than the male. For -3279, GD patients with higher TSH level or/and lower TRAb level were more frequent to carry A allele. We further analyzed the haplotypes of three SNPs and found that the haplotype CCA played a protective role in the susceptibility to GD. In contrast, the CAA and TCA haplotypes were associated with an increased susceptibility to GD. In addition, the mutation from C to A at the position of -3279 reduced the relative luciferase activity of Foxp3 promoter and decreased the expression of Foxp3 in GD patients. Our findings indicated that Foxp3 functional polymorphisms and haplotypes in promoter were associated with the susceptibility to GD in Chinese Han population.

Expression of Th17/Tregs Axis Associated Regulatory Factors in Patients with Acute Graft-Versus-Host Disease

Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the most valid methods for the treatment of malignant hematological diseases. Graft-versus-host disease (GVHD)，which remains the main complication after allo-HSCT，leads to the high transplant-related mortality. GVHD occurs as a result of T cell activation. Acute GVHD (aGVHD) is suggested to be predominantly related to T helper 1 (Th1) responses. Recent investigations have discovered the possible role of Th17 cells and regulatory T cells (Tregs) in the development of aGVHD. Th17 cells are found to have a direct role in the development of aGVHD, and adoptive transfer of in vitro-differentiated Th17 cells is capable of inducing lethal aGVHD. Clinical and experimental studies have suggested that Tregs could prevent and treat aGVHD, while preserve graft-versus-tumor activity. However, the controls of Th17/Tregs axis on the occurrence and development of aGVHD are not fully understood. The aim of this study is to investigate the expression levels and clinical significance of Th17/Tregs axis associated regulatory factors in patients with aGVHD.Methods The expression levels of Th17/Tregs axis associated regulatory factors (IL-17A, IL-23R, RORc, STAT-1, STAT-3, Foxp3, CD25, CTLA-4, GITR and TLR8) were analyzed in peripheral blood from 20 patients with aGVHD at the following two time points (at the onset of aGVHD and two weeks after the treatment), using real-time reverse transcription polymerase chain reaction with SYBR Green I staining. Fifteen patients responded effectively to the treatment within two weeks, and 5 did not respond within two weeks. Twenty healthy donors were selected for the control. The β2-microglobulin gene was used as an endogenous reference, and the relative mRNA expression level of each gene was evaluated by the 2-ΔCt×100% method.Results The expression levels of RORc, STAT-1 and STAT-3 genes in the untreated patients with aGVHD were significantly higher than that in the healthy donors (P<0.001, P<0.001 and P<0.001), while the expression levels of IL-17A and IL-23R genes were similar between the untreated patients with aGVHD and the healthy donors (P=0.147, P=0.190). The expression levels of GITR and TLR8 genes in the untreated patients with aGVHD were also significantly higher than that in the healthy donors (P=0.003, P<0.001). The expression levels of Foxp3, CD25 and CTLA-4 genes were similar between the untreated aGVHD patients and the healthy donors (P=0.298, P=0.934 and P=908). The expression level of TLR8 gene in the patients with aGVHD was significantly decreased after effective treatment (P=0.035).The expression levels of IL-17A, IL-23R, RORc, STAT-1, STAT-3, Foxp3, CD25, CTLA-4 and GITR genes in untreated patients with aGVHD had no significant change after effective treatment (P=0.778, P=0.875, P=0.730, P=0.177, P=0.925, P=0.397, P=0.778, P=0.470 and P=0.638). The expression level of Foxp3 gene was significantly decreased after ineffective treatment (P=0.043). The expression levels of IL-17A, IL-23R, RORc, STAT-1, STAT-3, CD25, CTLA-4, GITR and TLR8 genes were similar in patients at aGVHD onset and after ineffective treatment (P=0.686, P=0.500, P=0.686, P=0.345, P=0.225, P=0.345, P=0.345, P=0.893 and P=0.345).Conclusions Th17/Tregs axis associated regulatory factors might influence in the occurrence and development of aGVHD． DisclosuresXuan:It was supported by National Natural Science Foundation of China (81270647, 81300445, 81200388); National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding. Wu:It was supported by National Natural Science Foundation of China (81270647, 81300445, 81200388); National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding; It was supported by the Technology Plan of Guangdong Province of China (2012B031800403); the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding. Liu:It was supported by National Natural Science Foundation of China (81270647, 81300445, 81200388); National High Technology Research and Development Program of China (863 Program) (2011AA020105); National Public Health Grand Research Foundation (201202017): Research Funding; It was supported by Natural Science Foundation of Guangdong Province (S2012010009299); the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1);: Research Funding; It was supported by the Technology Plan of Guangdong Province of China (2012B031800403); the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding.

Differences in the Phenotype, Cytokine Gene Expression Profiles, and In Vivo Alloreactivity of T Cells Mobilized with Plerixafor Compared with G-CSF

Plerixafor (Mozobil) is a CXCR4 antagonist that rapidly mobilizes CD34(+) cells into circulation. Recently, plerixafor has been used as a single agent to mobilize peripheral blood stem cells for allogeneic hematopoietic cell transplantation. Although G-CSF mobilization is known to alter the phenotype and cytokine polarization of transplanted T cells, the effects of plerixafor mobilization on T cells have not been well characterized. In this study, we show that alterations in the T cell phenotype and cytokine gene expression profiles characteristic of G-CSF mobilization do not occur after mobilization with plerixafor. Compared with nonmobilized T cells, plerixafor-mobilized T cells had similar phenotype, mixed lymphocyte reactivity, and Foxp3 gene expression levels in CD4(+) T cells, and did not undergo a change in expression levels of 84 genes associated with Th1/Th2/Th3 pathways. In contrast with plerixafor, G-CSF mobilization decreased CD62L expression on both CD4 and CD8(+) T cells and altered expression levels of 16 cytokine-associated genes in CD3(+) T cells. To assess the clinical relevance of these findings, we explored a murine model of graft-versus-host disease in which transplant recipients received plerixafor or G-CSF mobilized allograft from MHC-matched, minor histocompatibility-mismatched donors; recipients of plerixafor mobilized peripheral blood stem cells had a significantly higher incidence of skin graft-versus-host disease compared with mice receiving G-CSF mobilized transplants (100 versus 50%, respectively, p = 0.02). These preclinical data show plerixafor, in contrast with G-CSF, does not alter the phenotype and cytokine polarization of T cells, which raises the possibility that T cell-mediated immune sequelae of allogeneic transplantation in humans may differ when donor allografts are mobilized with plerixafor compared with G-CSF.

Enhancement Of Human Regulatory γδ T Cells In Vitro Induced By Granulocyte Colony-Stimulating Factor

Background and ObjectiveGraft versus host disease (GVHD) is the main complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Regulatory γδ T cells (γδ Tregs), which express Foxp3 and primarily belong to CD27+CD25high phenotype, are a novel subset of cells with immunosuppressive function and have the potential application prospect in GVHD therapy. Peripheral blood mononuclear cells (PBMCs) could be induced to generate γδ Tregs in vitro by stimulating with anti-TCR γδ and transforming growth factor-β (TGF-β). Our previous studies demonstrated that granulocyte colony-stimulating factor (G-CSF) had immunomodulatory effect on T cells; G-CSF mobilization influenced the distribution and clonality of TRGV and TRDV repertoire (T cell receptors of γδ T cells), and significant positive correlation was observed between the invariable clonality of TRDV1 gene repertoire after G-CSF mobilization and low incidence of GVHD in recipients. To further characterize the immunoregulatory functions of G-CSF, this study explored the possibility of γδ Tregs induced by G-CSF in vitro. MethodsPBMCs of healthy donors were cultured in vitro by stimulating with anti-TCR γδ and different cytokines for 9-12 days. The culture system was grouped based on the difference of added cytokines, including: (1) TGF-β (2) G-CSF (3) TGF-β+G-CSF (4) blank group. The induced cells were used as effector cells in the carboxylfluorescein diacetate succinimidyl ester (CFSE) assays and cell immunophenotyping was analyzed by flow cytometry. Autologous CD4+T cells were purified by microbeads, labeled with CFSE, used as responder cells, and finally co-cultured with effector cells. After 5 days incubation, cells were harvested and analyzed for flow cytometry by gating on the CFSE-labeled cells. The expression levels of Foxp3 and CD25 genes were detected by real-time polymerase chain reaction. ResultsAfter 9 days' culture in vitro, the proportion of Foxp3+ γδ T cells was 7.87% in the blank group, which was significantly lower than that in the TGF-β, G-CSF and TGF-β + G-CSF group (62.3%, 52.9% and 63.5%) (P<0.001). The proportions of CD25+γδ T and CD27+γδ T cells were 3.9% and 3.5% in the blank group, which were also significantly lower than that in the TGF-β, G-CSF and TGF-β + G-CSF group (CD25+γδ T cells: 43.5%, 43.4% and 44.4%; CD27+γδ T cells: 41.5%, 41.9% and 44.0%, respectively) (P<0.001, P<0.001). The effector cells induced by anti-TCR γδ with TGF-β, G-CSF and TGF-β + G-CSF all manifested a more significant suppressive effect on responder cells than cells induced only by anti-TCR γδ. However, there was no significant difference in the effect of cell proliferation suppression in the TGF-β, G-CSF and TGF-β + G-CSF groups. The expression level of Foxp3 gene was 0.384% in the blank group, which was significantly lower than that in the TGF-β, G-CSF and TGF-β + G-CSF group (0.623%, 0.639% and 0.843%, respectively) (P<0.001). The expression level of Foxp3 in TGF-β+ G-CSF group was significantly higher than that in the TGF-β and G-CSF group (P=0.001, P=0.007). The expression level of CD25 gene was similar among the four groups (P=0.457). ConclusionsThe generation of γδ Tregs could be enhance in vitro induced by G-CSF. This may be one of the reasons that G-CSF play an immunoregulatory role on decreased GVHD onset during allo-HSCT. Disclosures:Wu:National Natural Science Foundation of China (No. 81200388); the Technology Plan of Guangdong Province of China (No. 2012B031800403); the project of the Zhujiang Science & Technology Star of Guangzhou city (No. 2013027).: Research Funding.

CD4 + CD25 + regulatory T cells-induced tolerance of islet transplantation by targeting OX40/ OX40L costimulation pathway

Objective To observe the role of OX40/OX40L costimulation pathway in the CD4+CD25 + regulatory T (Treg) cells-induced tolerance of islet transplantation.Methods Diabetes mellitus was induced in C57BL/6 mice as recipients,and islets from DBA/2 mice were transplanted into the C57BL/6 mice.The recipients were divided into three groups (n =8):treated with IgG as controls,antiOX40L mAb (RM134L) + anti-CD154 mAb (MR1),and RM134L + MR1 + anti-CD25 mAb.The mean survival time (MST) of allograft was observed.The expression of OX40 in T effector cells,Treg cells of wide type and CD154 knock-out mice was detected.The expression of Foxp3 gene in allograft in the absence and absence of anti-OX40L mAb and anti-CD154 mAb was detected.T effector cells and Treg cells were co-cultured at different ratio,and the cell proliferation was assayed.Results The MST in RM134L +MR1 group was ＞ 150 days (n =8),significantly longer than in IgG group ( MST + 19 days,n =5 ) and RM134L + MR1 + anti-CD25 mAb (23 days,n =4) (P ＜0.05).Anti-OX40L mAb and anti-CD154 mAb treatment could successfully induce tolerance,and also induce donor specific tolerance,but the tolerance was blocked by anti-CD25 mAb treatment (P ＜ 0.05 ).OX40 was expressed on the Treg cells surface,whereas the T effector cells had no OX40 expression.The expression level of Foxp3 gene in the allograft treated with anti-OX40L mAb and anti-CD154 mAb was 123 AU,significantly higher than without treatment (21 AU) and syngenic control ( 8 AU) ( P ＜ 0.05 ).After Treg cells were co-cultured with T effector cells at different ratio ( 1∶2 and 1∶ 4),the mean count per minute (CPM) of T effector cells was 63 x 103and 41 × 103 respectively (P ＜0.05),and after addition of OX40,the CPM was 128 × 103 and 135 × 103respectively ( P ＞ 0.05 ).Conclusion CD4 + CD25 + Treg cells were critical to tolerance induction in mice islet transplant recipients through targeting OX40/OX40L costimulation pathway. Key words: Islet transplantation; Regulatory T cells; Tolerance; OX40

The in vitro effect of oxidized LDL and PHA on proliferation and gene expression of regulatory T cells in patients with atherosclerosis.

Atherosclerosis is a chronic inflammatory condition that affects the arterial wall. Oxidized low-density lipoprotein (ox-LDL) seems to have an important role in atherosclerotic plaque formation.This study was performed to investigate the effects of ox-LDL as well as PHA on proliferation and gene expression of peripheral blood mononuclear cells (PBMCs) in patients with atherosclerosis compared to healthy controls. Proliferation of PBMCs was assessed by BrdU assay, while gene expression was assessed by real-time PCR. Both PHA and ox-LDL significantly induced proliferation of PBMCs of patients and controls. PBMCs from controls showed significantly higher proliferation when stimulated with ox-LDL compared to patients. Expression of TGF-β was significantly lower in PBMCs from patients compared to healthy controls (p<0.001). Following simulation with PHA, TGF-β and Foxp3 gene expression levels in patients and controls were significantly decreased (p<0.001). Expression of Foxp3 in PBMCs treated with ox-LDL was significantly decreased in patients and controls.Decreased expression of TGF-β and Foxp3 genes after ox-LDL stimulation may be due to more sensitivity of Treg cells than effector T cells to ox-LDL. Presence of ox-LDL within atheroma could be associated with the diminished population of Treg cells in the atherosclerotic patients.

The Expression of GATA-3 and FoxP3 Genes and the Frequency of Tregs Are Correlated with Gvhd Following Allogeneic HSCT

AbstractAbstract 5181 ObjectiveHematopoietic stem cell transplantation (HSCT) is a common therapeutic option for leukemia, but its use is limited by mortality due to graft-versus-host-disease (GVHD). Naturally occurring regulatory T cells (Tregs) are mediators of immunologic tolerance that attenuate GVHD in experimental models. Tregs were first defined by a CD4+CD25+ phenotype, but subsequent studies identified forkhead box protein 3 (FoxP3) as a highly specific marker in both mouse and human T cells with regulatory function. GATA-3 is a member of the GATA family of transcription factors, and is sufficient to induce a Th2 phenotype. GATA-3 could act as an inhibitor of FoxP3 expression in early T cell differentiation was found in transgenic mice model recently. But whether GATA-3 expression could correlate with GVHD incidence, or whether GATA-3 could influence Tregs recovery and contribute to the development of GVHD are still not clear. So we investigated the frequency of Tregs in the early period after HSCT and at the time of GVHD onset, and quantitatively measured the expression of GATA-3 and FoxP3 at GVHD onset for analyzing the correlation to the development of GVHD. MethodsSixty-seven leukemia patients underwent allogenic (allo-) HSCT were enrolled in this investigation, including 45 males and 22 females (median age: 32.3 years, range 21≂f45). Fourteen healthy individuals (7 males and 7 females, median age: 30.3 years, range 26≂f40) served as controls. Frequencies of T lymphocyte subsets were determined by flow cytometry. The expression levels of human FoxP3 and GATA-3 genes were determined by real-time quantitative PCR with SYBR Green I technique. ResultsThe frequencies of CD4+ CD25+ T cells were lower at week 4 post-HSCT, but significant higher after week 4 and lower again after week 8 (P=0.000). The frequencies of FoxP3+ T cells were also lower at week 4 and remained low levels within 12 weeks after HSCT. As early as 4 to 8 weeks after HSCT, the frequencies of CD4+ FoxP3+ T cells were lower than that of week 4 (P=0.048), but the frequencies of CD25+ FoxP3+ T cells were higher than that of week 4 (P=0.022).Frequencies of CD4+ CD25+ cells in patients at GVHD onset were similar in patients without GVHD (P=0.262) or healthy individuals (P=0.166). But the frequencies of FoxP3+ cells, FoxP3+ CD25+ cells and FoxP3+ CD4+ cells in patients at GVHD onset were significantly lower than that in patients without GVHD (P=0.000, 0.004, and 0.001). The FoxP3 gene expression levels in patients at GVHD onset were significantly lower than that in patients without GVHD (P=0.044), but the GATA-3 gene expression levels were significantly higher in patients at GVHD onset compared to patients without GVHD (P=0.005). The FoxP3 gene expression levels in patients at aGVHD onset were significantly higher than that in patients at cGVHD onset (P=0.038), but the GATA-3 gene expression levels were significantly lower in patients at aGVHD onset compared to patients at cGVHD onset (P=0.004).A highly significant correlation between FoxP3 and GATA-3 genes expression levels existed in healthy individuals (Spearman correlation coefficient r=-0.893, P=0.007). Correlations between FoxP3 and GATA-3 genes expression levels were also significant in patients at aGVHD onset (Pearson correlation coefficient r=0.749, P=0.020) or cGVHD onset (Pearson correlation coefficient r=0.538, P=0.071) or in GVHD (Spearman correlation coefficient r=0.370, P=0.090). But there is no significant correlation between FoxP3 and GATA-3 genes expression levels in patients without GVHD (Pearson correlation coefficient r=-0.141, P=0.821). ConclusionsGATA-3 and FoxP3 genes expression and the frequency of Tregs are correlated with GVHD following allogeneic HSCT. GATA-3 can influence the expression of FoxP3 in the development of GVHD.Supported by National Natural Science Foundation of China(30971300), Science and Technology Planning Project of Guangdong Province of China (2009A030200007) and China Postdoctoral Science Foundation (200902332, 20080440776). Disclosures:No relevant conflicts of interest to declare.

Functional Analysis and Gene Expression Profile of Umbilical Cord Blood Regulatory T Cells.

Abstract 4085Poster Board III-1020 IntroductionUmbilical cord blood (CB) stem cells are now broadly used in the unrelated stem cell transplant setting and comparative studies with different stem cell sources have shown that CB transplant is characterized by a lower risk of graft-versus-host disease (GVHD). The immaturity of CB T cells has been generally regarded as the main contributing factor accounting for this phenomenon; the possible role played by CB regulatory T cells (Tregs) for the suppression of the allogeneic T-cell response is now under investigation, but very scare data are so far available. Aim of this study was to analyze and compare the functional properties and the gene expression profile of Tregs expanded from CB units with those expanded from the peripheral blood (PB) of adult normal donors. MethodsTregs were purified from mononuclear cells obtained from 23 CB units and from the PB of 13 adult normal donors using the CD4+CD25+ regulatory T-cell isolation kit (Miltenyi Biotec) and expanded for 6 days in 96-well U-Bottom plates coated with the anti-CD3 (5 ug/ml) and anti-CD28 (5 ug/ml) MoAbs in the presence of IL-2 (100 U/ml). Immunophenotypic analyses were performed before and after expansion. To assess their suppressive functions, expanded Tregs were seeded with autologous effector T cells stimulated with allogeneic dendritic cells (DC) pulsed with apoptotic leukemic blasts, then incubated with [3H]-thymidine and counted in a beta-counter. Suppressor activity was measured as [3H]-thymidine incorporation in the presence or absence of Tregs. The IL-10 production capacity of expanded Tregs was tested using an ELISA assay. The two-sided student t test was used to evaluate the significance of differences between groups. Gene expression profile experiments were performed using the HGU133 Plus 2.0 arrays (Affymetrix); statistical analyses were carried out using the dChip software; a t test was used to evaluate the presence of specifically expressed classes of genes. Functional annotation analysis was performed using the DAVID software. ResultsCB and PB Tregs presented similar immunophenotypic appearances before and after expansion. Im particular, after expansion they presented a comparable expression of surface CD4, CD25, CD62L, CCR5 and CD45RO, and of cytoplasmic CTLA-4 and Foxp3, while they both were negative for the CD45RA antigen, thus indicating the loss of their naïve features. On the contrary, Tregs obtained from CB (n=23) presented a much higher expansion capacity compared to those obtained from PB (n=13): mean fold increase (range), CB 10.3 (1.6-24), PB 3.9 (1.5-10), p 0.003. CB expanded Tregs (n=6) exerted a potent suppressive function on the proliferative reaction of T cells stimulated by allogeneic DC, that resulted inferior even though not significantly compared to that exerted by PB expanded Tregs (n=5): mean fold reduction (range), CB 7.8 (2.5-15.1), PB 14.3 (1.5-23.7), p 0.14. Tregs expanded from CB (n=4) and PB (n=1) presented a high and comparable in vitro IL-10 production capacity: mean pg/ml (range), CB 326.5 (226-426), PB 382. Gene expression profile analysis showed a higher number of upregulated genes in Tregs expanded from CB (n=2) compared to Tregs expanded from PB (n=3); among them, a significant enrichment of genes involved in cell proliferation, cell cycle checkpoints, signal transduction, cell differentiation, apoptosis, TGF-β receptor pathway and the GrNH pathway was observed. This suggests that CB Tregs retain a more undifferentiated program and are characterized by the high expression of genes which might provide an advantage in cell expansion. Finally, when looking at the Foxp3 gene expression levels, no difference was observed between the two populations. ConclusionsThese results demonstrate that Tregs contained in CB retain an expansion potential superior to that of Tregs isolated from the PB of normal donors, as confirmed by functional analyses and gene profile. Tregs expanded from CB and PB seem to exert a potent and comparable suppressive function of the proliferative effect in mixed lymphocyte reaction assays. The maintaining of the modulatory properties after expansion is confirmed by the expression of the Foxp3 gene and protein, and by the production of IL-10. These data offer further insights into the understanding of the biology of CB transplantation indicating a possible role played by CB Tregs in the suppression of the allogeneic T-cell response. Disclosures:No relevant conflicts of interest to declare.