Unconventional cytokine profiles and development of T cell memory in long-term survivors after cancer vaccination

Cancer vaccine trials frequently report on immunological responses, without any clinical benefit. This paradox may reflect the challenge of discriminating between effective and pointless immune responses and sparse knowledge on their long-term development. Here, we have analyzed T cell responses in long-term survivors after peptide vaccination. There were three main study aims: (1) to characterize the immune response in patients with a possible clinical benefit. (2) To analyze the long-term development of responses and effects of booster vaccination. (3) To investigate whether the Th1/Th2-delineation applies to cancer vaccine responses. T cell clones were generated from all nine patients studied. We find that surviving patients harbor durable tumor-specific responses against vaccine antigens from telomerase, RAS or TGFβ receptor II. Analyses of consecutive samples suggest that booster vaccination is required to induce robust T cell memory. The responses exhibit several features of possible clinical advantage, including combined T-helper and cytotoxic functionality, recognition of naturally processed antigens and diverse HLA-restriction and fine-specificity. CD4⁻CD8⁻ T cell clones display unconventional cytotoxicity and specifically kill tumor cells expressing mutated TGFβ receptor II. Cytokine profiling on the long-term survivors demonstrates high IFNγ/IL10-ratios, favoring immunity over tolerance, and secretion of multiple chemokines likely to mobilize the innate and adaptive immune system. Interestingly, these pro-inflammatory cytokine profiles do not follow a Th1/Th2-delineation. Most IFNγ^high/IL4^low/IL10^low cultures include high concentrations of hallmark Th2-cytokines IL-5 and IL-13. This does not reflect a mixture of Th1- and Th2-clones, but applies to 19/20 T cell clones confirmed to be monoclonal through TCR clonotype mapping. The present study identifies several factors that may promote clinical efficacy and suggests that cytokine profiling should not rely on the Th1/Th2-paradigm, but assess the overall inflammatory milieu and the balance between key cytokines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Immunofluorescence localization of the regulatory subunit type II of cAMP-dependent protein kinase in PC12 and 3T3 cells in different proliferative states

Localization of the regulatory subunit of cAMP-dependent protein kinase type II was studied in proliferating and quiescent fibroblasts 3T3 and in a cell line of neural origin pheochromocytoma PC12. In actively proliferating PCl2 cells the regulatory subunit was found to be localized in the nucleus. Transition of these cells into a quiescent state was accompanied by a regulatory subunit translocation to the cytoplasm. In 3T3 cells the regulatory subunit was localized in the cytoplasm both in the quiescent and proliferating (though less actively than PC12 cells) states. Similar results were obtained both with monoclonal antibodies and with rabbit monospecific antiserum raised against the regulatory subunit type II from pig brain.     

Age-associated parallel increase of Foxp3CD4 regulatory and CD44CD4 memory T cells in SJL/J mice

Effector/memory T cells (Tem) are required to maintain successful immunity, while regulatory T cells (Treg) are required to prevent excessive/uncontrolled inflammation and/or autoimmunity. Although both Tem and Treg cells are increased during aging, the relationship between the increased proportion of Foxp3⁺ Treg cells and CD44⁺ Tem cells with aging is not clearly understood. We found in this report that Foxp3⁺ Treg cells are increased in parallel with CD44⁺ Tem cells in SJL/J mice with aging, and that all Foxp3⁺ Treg cells are of CD44⁺ Tem phenotype, suggesting that the increased Foxp3⁺ Treg cells originated from the expanded pool of CD44⁺ Tem cells with aging. Our in vitro kinetic studies further suggested that Foxp3⁺ Treg cells are converted through the CD44⁺ stage. Furthermore, we observed that although the balance between Foxp3⁺ Treg and CD44⁺Foxp3⁻ Tem cells remained with aging, the aged mice have higher ratios of both Tem and Treg cells vs. naïve T cells resulting in the “shrunken” naïve T cell pools. Our results suggest that an age-associated imbalance of T cell repertoire is a mechanism that contributes to spontaneous occurrence of Hodgkin’s-like lymphoma in aged SJL/J mice.     

I kappa B kinase alpha (IKKα) activity is required for functional maturation of dendritic cells and acquired immunity to infection

Dendritic cells (DC) are required for priming antigen‐specific T cells and acquired immunity to many important human pathogens, including Mycobacteriuim tuberculosis (TB) and influenza. However, inappropriate priming of auto‐reactive T cells is linked with autoimmune disease. Understanding the molecular mechanisms that regulate the priming and activation of naïve T cells is critical for development of new improved vaccines and understanding the pathogenesis of autoimmune diseases. The serine/threonine kinase IKKα (CHUK) has previously been shown to have anti‐inflammatory activity and inhibit innate immunity. Here, we show that IKKα is required in DC for priming antigen‐specific T cells and acquired immunity to the human pathogen Listeria monocytogenes. We describe a new role for IKKα in regulation of IRF3 activity and the functional maturation of DC. This presents a unique role for IKKα in dampening inflammation while simultaneously promoting adaptive immunity that could have important implications for the development of new vaccine adjuvants and treatment of autoimmune diseases.     

慢病毒介导的小鼠PINK1基因RNAi载体的构建及在NIH3T3细胞中的筛选

目的:构建筛选靶向特异PINK1-siRNA慢病毒表达载体,感染小鼠胚胎细胞(NIH3T3)验证该病毒载体的敲减效率,为研究帕金森病的发病机制奠定基础。方法:构建2对靶向小鼠PINK1-siRNA序列(KD1和KD2),将这2对序列连接在GV118上,将重组载体和病毒包装的辅助质粒共转染293 T细胞,获得慢病毒颗粒,再将该病毒颗粒转染入NIH3T3细胞,用qRT-PCR验证细胞内PINK1 mRNA表达水平以验证敲减效果。结果:筛选出可以用于后续实验的高效靶向KD2序列,并成功转染到小鼠NIH3T3细胞中,在感染复数(MOI)为50时,KD2的沉默效果最好,敲减率达到66.4%。结论:成功构建了高效靶向小鼠PINK1-siRNA慢病毒载体,其可稳定转染小鼠NIH3T3细胞,可高效抑制PINK1 mRNA的表达。为下一步利用其感染神经细胞或注入动物脑内,进一步研究PINK1基因在帕金森病发病过程中的作用环节和机制提供了分子生物学的技术基础。     

Characterization of T cell clones specific to a determinant of hepatitis B virus core and e antigens in chronic type B hepatitis: Implication for a T cell mechanism of HBV immunopathogenesis

T cell clones specific for hepatitis B core (HBcAg) and e (HBeAg) antigens of hepatitis B virus (HBV) were generated from liver infiltrates of HBeAg-positive patients. Analyzed with a panel of overlapping synthetic peptides spanning the complete sequences of HBcAg and HBeAg, eight clones responded specifically to the e2 peptide (PAYRPPNAPIL; amino acid residues 130–140 of HBcAg and HBeAg), which was doubly restricted by class I and II molecules. A preferential usage of the T cell receptor (TCR) chain variable (V) gene was found: V12.1 for five HLA-Cw9(3)-restricted cytotoxic T lymphocyte (CTL) clones, and V7.1 for three other HLA-DRw52-restricted type 1 helper T cell (Th1) clones. Although heterogeneous in the usage of TCR chain joining region (J) segments, their junctional-region sequences revealed conserved hydrophilic serine residues in seven of the eight e2-specific T cell clones. Single alanine substitution of the centrally located and the only hydrophilic asparagine residue of e2 peptide abrogated T cell responsiveness. The nonstimulatory e2 analogue could competitively inhibit e2-specific responses. These results demonstrate that both CTL and Th1 clones recognizing a determinant of HBcAg and HBeAg are present in the liver of chronic hepatitis B patients. The preferential V gene usage and the expression of conserved residues in junctional-region sequences of TCR chains by viral-peptide-specific, intrahepatic T cells may provide a T cell mechanism of HBV immunopathogenesis.     

S100A6 (calcyclin) deficiency induces senescence‐like changes in cell cycle,morphology and functional characteristics of mouse NIH 3T3 fibroblasts

S100A6 (calcyclin) is a calcium binding protein with two EF‐hand structures expressed mostly in fibroblasts and epithelial cells. We have established a NIH 3T3 fibroblast cell line stably transfected with siRNA against S100A6 to examine the effect of S100A6 deficiency on non‐transformed cell physiology. We found that NIH 3T3 fibroblasts with decreased level of S100A6 manifested altered cell morphology and proliferated at a much slower pace than the control cells. Cell cycle analysis showed that a large population of these cells lost the ability to respond to serum and persisted in the G0/G1 phase. Furthermore, fibroblasts with diminished S100A6 level exhibited morphological changes and biochemical features of cellular senescence as revealed by β‐galactosidase and gelatinase assays. Also, S100A6 deficiency induced changes in the actin cytoskeleton and had a profound impact on cell adhesion and migration. Thus, we have shown that the S100A6 protein is involved in multiple aspects of fibroblast physiology and that its presence ensures normal fibroblast proliferation and function. J. Cell. Biochem. 109: 576–584, 2010. © 2009 Wiley‐Liss, Inc.     

Differential requirements for CD80/86-CD28 costimulation in primary and memory CD4 T cell responses to vaccinia virus

Vaccinia virus infection can confer immunity to smallpox by inducing potent T cell and antibody responses. While the CD8 T cell response to vaccinia virus has been well characterized, less is known about factors required for priming and memory for the CD4 T cells. Focusing on two recently described epitopes, we show that after intranasal infection, both I1L and L4R epitopes are co-dominant during the acute response, but the I1L epitope dominates during memory. CD4 T cell priming was intact in the absence of CD80/86, however secondary responses were reduced. This contrasts with our previous data showing CD80/86–CD28 interaction is required for optimal primary and memory CD8 T cell responses. The absence of CD80/86 also changed the immunodominance hierarchy during memory, with the I1L and L4R responses becoming co-dominant in knockout mice. These data highlight different costimulatory requirements for primary CD4 and CD8 T cell responses to vaccinia virus.     

Optimal conditions for blastogenic response of peripheral blood lymphocytes to T and B cell mitogens in cynomolgus monkeys

Optimal conditions for the mitogen-induced proliferation of T and B lymphocytes of cynomolgus monkeys were determined. The T cell mitogens concanavalin A and phytohemagglutinin, at concentrations of 1.25–10 μg/ml and 1.25–10 μg/ml, respectively, and the T and B cell mitogen pokeweed mitogen, at concentrations of 0.2–10 μg/ml, induced high lymphoproliferative responses, the average stimulation index (SI) being 34–93. Since suitable mitogens have not been reported for monkey B cells, we tested three types of lipopolysaccharide (LPS): two derived from Escherichia coli—one extracted with phenol and one extracted with trichloroacetic acid (TCA); and one derived from Salmonella typhimurium, extracted with phenol. All three LPS had a high mitogenic effect for monkey lymphocytes, with SI of 2.3–6.4. The highest response was observed for 25 μg/ml of Salmonella LPS, and the addition of trypsin to the culture augmented the proliferative response of low or non-responder lymphocytes. © 1994 Wiley-Liss, Inc.     

柚皮苷对高糖作用下MC3T3-E1细胞活力和Akt通路相关因子表达的影响

目的探讨柚皮苷（NG）对高糖环境下MC3T3-E1细胞活力的影响及可能的分子机制。 方法体外培养小鼠MC3T3-E1细胞，实验分5组：对照组（正常无血清培养基）、高糖组（含25 mmol/L葡萄糖）、0.1 μmol/L +高糖组（0.1 μmol/L NG + 25 mmol/L葡萄糖）、1 μmol/L +高糖组（1 μmol/L NG+25 mmol/L葡萄糖）、10 μmol/L +高糖组（10 μmol/L NG+25 mmol/L葡萄糖）。药物干预后，采用CCK-8法检测细胞活力；实时荧光定量PCR（qPCR）法检测细胞成骨特异性转录因子（Runx2）、胰岛素样生长因子-1（IGF-1）、蛋白激酶（Akt1）mRNA的表达；蛋白质印迹法（Western blot）检测细胞碱性磷酸酶（ALP）、Akt1、IGF-1蛋白的表达。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。 结果CCK8检测结果显示，与对照组比较，高糖组细胞OD值（12 h：0.90±0.01比0.80±0.01，24 h：1.00±0.05比0.84±0.01，48 h：1.09±0.03比0.90±0.01）均降低，差异有统计学意义（P < 0.01）；与高糖组比较，0.1 μmol/L+高糖组细胞OD值（24 h：0.84±0.01比0.93±0.05，48 h：0.90±0.01比0.99±0.01）、1 μmol/L +高糖组和10 μmol/L+高糖组OD值（12 h：0.80±0.01比0.92±0.01、1.01±0.32，24 h：0.84±0.01比1.01±0.04、1.16±0.03，48 h：0.90±0.01比1.12±0.02、1.20±0.02）均升高，差异有统计学意义（P < 0.05）。与对照组比较，高糖组细胞Runx2、IGF-1、Akt1的mRNA的表达水平（24 h：1.00比0.34±0.02、1.00比0.34±0.01、1.00比0.15±0.02）、（48 h：1.00比0.72±0.03、1.00比1.09±0.07、1.00比0.38±0.04）降低，差异有统计学意义（P < 0.01）。与高糖组比较，1 μmol/L +高糖组和10 μmol/L+高糖组细胞Runx2、IGF-1、Akt1的mRNA表达水平（24 h：0.34±0.02比0.62±0.09、0.64±0.05，0.34±0.01比0.77±0.03、1.02±0.07，0.15±0.02比0.24±0.08、0.4±0.09）、（48 h：0.72±0.03比1.27±0.02、1.37±0.02，1.09±0.07比2.44±0.19、2.73±0.04，0.38±0.04比0.86±0.06、1.43±0.03）均升高，差异有统计学意义（P < 0.05）。与对照组比较，高糖组细胞ALP、Akt1、IGF-1蛋白表达水平（48 h：1.00比0.72±0.02、1.00比0.89±0.03、1.00比0.09±0.01）均降低，差异有统计学意义（P < 0.05）；与高糖组比较，0.1 μmol/L+高糖组、1 μmol/L+高糖组和10 μmol/L+高糖组ALP、Akt1、IGF-1蛋白表达水平（48 h：0.72±0.02比1.92±0.02、2.30±0.30、3.09±0.10，0.89±0.03比1.50 ± 0.03、1.43±0.04、1.40±0.13，0.09±0.01比1.75±0.01、2.30±0.31、2.07±0.07）均升高，差异有统计学意义（P < 0.05）。 结论NG逆转高糖诱导的MC3T3-E1细胞活力减退；同时改善高糖的抑制作用，促进MC3T3-E1细胞IGF-1、AKt-1、Runx2 mRNA和IGF-1、AKt-1、ALP蛋白的表达。     

Evaluation of CD4+CD25+FoxP3+ T cell populations,IL-10 production,and their correlation with clinical and biochemical parameters in sickle cell anemia patients with leg ulcers

Leg ulcers (LUs) are a debilitating complication of sickle cell anemia (SCA), with inflammation known to play a crucial role in their pathogenesis. Many studies have described the roles of T helper type 1 (Th1) and Th2 pathways in SCA; however, defects in anti-inflammatory responses are poorly understood. We evaluated interleukin (IL)-10 levels in serum and peripheral blood mononuclear cells (PBMCs) in SCA patients with leg ulcers (SCALU) and without leg ulcers (SCAWH) in addition to CD4⁺ CD25⁺FoxP3⁺ T cell populations and their its IL-10 expression. In stimulated and unstimulated PBMC cultures, SCALU patients produced higher levels of IL-10 than those in the SCAWH group. Higher levels of IL-10 in SCALU patients correlated with a history of osteonecrosis in stimulated and unstimulated cultures when compared with those in SCAWH. Immunophenotyping revealed that SCALU patients had a higher proportion of CD4⁺CD25⁺FoxP3⁺, Tr1 and CD4⁺CD25⁺FoxP3⁺IL-10⁺ T cells than other groups. Our findings revealed that IL-10 levels were increased in unstimulated cells from the SCALU group, and that this group also presented with a predominant CD4⁺ CD25⁺FoxP3⁺ cell population despite many of those cells being IL-10 negative.     

The effect of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline on cell growth, cell cycle, induction of DNA fragmentation, and activity of caspase 3 in murine leukemia L1210 cells and fibroblast NIH-3T3 cells

Quinazolines are multitarget agents, which have broad spectrum of biological activity, and some of them are now in cancer clinical testing. 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline is a new synthetically prepared derivative, which in our previous study showed cytotoxic effects on cancer cell lines HeLa and B16. Quinazoline, at micromolar concentrations, induced morphological changes and necrosis of B16 cells, and at nanomolar concentrations it produced changes of F-actin cytoskeleton. It did not cause changes in the cell cycle, did not induce apoptotic cell death in B16 cells, did not have a mutagenic effect, and did not even behave as a typical intercalating agent. Little significant reduction of tumor volume in intramuscular transplanted B16 cells was observed. The aim of the present study was to examine the cytotoxic effect of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline on murine leukemia L1210 cells and fibroblast NIH-3T3 cells. Induction of cell morphology and cell cycle changes, induction of apoptosis and caspase 3 activity were studied. Quinazoline acted cytotoxically on both cell lines. The sensitivity of leukemia L1210 cells to the quinazoline was higher than that of fibroblast NIH-3T3. The IC(100) was 12 microM for L1210 cells and 24 microM for NIH-3T3 cells. No effect of quinazoline on the cell cycle profile of L1210 and NIH-3T3 was detected, however, quinazoline induced an increase of the sub-G(0) cell fraction, apoptotic DNA fragmentation, and apoptotic morphological changes at a concentration of 12 microM. This quinazoline concentration induced caspase 3 activity. Our results demonstrated that induction of apoptotic cell death via activation of caspase 3 contributed to the cytotoxic effects of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline in murine leukemia L1210 cells.     

Human and viral membrane–associated E3 ubiquitin ligases MARCH1 and MIR2 recognize different features of CD86 to downregulate surface expression

Immune-stimulatory ligands, such as major histocompatibility complex molecules and the T-cell costimulatory ligand CD86, are central to productive immunity. Endogenous mammalian membrane-associated RING-CHs (MARCH) act on these and other targets to regulate antigen presentation and activation of adaptive immunity, whereas virus-encoded homologs target the same molecules to evade immune responses. Substrate specificity is encoded in or near the membrane-embedded domains of MARCHs and the proteins they regulate, but the exact sequences that distinguish substrates from nonsubstrates are poorly understood. Here, we examined the requirements for recognition of the costimulatory ligand CD86 by two different MARCH-family proteins, human MARCH1 and Kaposi''s sarcoma herpesvirus modulator of immune recognition 2 (MIR2), using deep mutational scanning. We identified a highly specific recognition surface in the hydrophobic core of the CD86 transmembrane (TM) domain (TMD) that is required for recognition by MARCH1 and prominently features a proline at position 254. In contrast, MIR2 requires no specific sequences in the CD86 TMD but relies primarily on an aspartic acid at position 244 in the CD86 extracellular juxtamembrane region. Surprisingly, MIR2 recognized CD86 with a TMD composed entirely of valine, whereas many different single amino acid substitutions in the context of the native TM sequence conferred MIR2 resistance. These results show that the human and viral proteins evolved completely different recognition modes for the same substrate. That some TM sequences are incompatible with MIR2 activity, even when no specific recognition motif is required, suggests a more complicated mechanism of immune modulation via CD86 than was previously appreciated.     

Regulation and specificity of MNDA expression in monocytes,macrophages, and leukemia/B lymophoma cell lines

The expression of the human myeloid cell nuclear differentiation antigen (MNDA) was observed specifically in cells of the granulocyte-macrophage lineage in our earlier reports. The specificity of MNDA expression for cells in the granulocyte-macrophage lineage was reexamined in cell line established from patients with philadelphia chromosome-positive chroni myeloid leukemia. Cell lines that expressed MNDA exhibited myeloid cell features and granulocyte or monocyte defferentiation could be induced in vitro, while cell lines exhibiting properties of very early stage cells of multipotential cells ded not express MNDA. Cells orginating from cases of burkitt's lymphoma were negative. By contrast, three Iymphoblastoid cell lines (immortalized in vitro with Epstein-Barr virus) were weakly positive and MNDA was up-regulated by interferon-α (IFN-α) treatment. As we reported previously, MNDA mRNA level in adherent monocytes is elevated by IFN-α; in this study, we further assessed MNDA expression in in vitro monocyte-derived macrophages. Three addditional agents (endotoxin, phytohemagglutinin, and photbol ester) and other conditions that affect function, cutokine production, defferentiation, and/of growth of monocytes were examined for their ability to alter MNDA expression. The results varied with the agent, cell type, and stage of differentiation. Changes in MNDA expression occurred slowly (hours to days), suggesting that MNDA could mediate changes realized over a long period. The results also reveal a discordance in certain MNDA Positiva cells between steady-state levels of changes in levels of protein and mRNA indicating that the regulation of MNDA expression occurs at more than one point. Changes in MNDA expression are consistent with a role in opposing macrophage defferentiation and activation of monocytes/macrophages.     

Differences in sialyl transferase activity and in concanavalin-A agglutination between T and B lymphoblastoid cell lines.

The concanavalin A agglutination patterns, sialyl transferase activity and sialic acid content were studied for cultured lymphoblastoid cell lines possessing either T or B surface markers. Concanavalin A caused marked agglutination of the two B cell lines studied, Raji and SB, while the two T cell lines, HSB-2 and CCRF-CEM, failed to agglutinate with this lectin. The surface sialyl transferase activity of the two B lines was significantly higher than on the two T lines studied. In contrast, the total cellular sialic acid content or the surface sialic acid that was released from the T and B cell lines by neuraminidase was not significantly different. This study indicates that T and B lymphoblastoid cells possess different levels of surface located sialyl transferase activity and display different agglutination patterns with Con A. Perhaps these assays can be of value in differentiating various classes of neoplastic lymphoid cells.     

Correlation between the high expression of C/EBPbeta protein in F442A cells and their relative resistance to antiadipogenic action of TCDD in comparison to 3T3-L1 cells

We compared the ability of two clonally derived murine preadipocyte cell lines, 3T3-L1(L1) and 3T3-F442A (F442A), to differentiate after treatment by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and found that the former cell line was clearly suppressed by TCDD but the latter was not. It was initially postulated that the easiest way to explain the lack of response to TCDD in F442A cells could be an alteration in aryl hydrocarbon receptor (AhR) functionality. This hypothesis was tested first, but no differences were found in the levels or functions of AhR. To find an alternate explanation for such a differential effect of TCDD, we tested the action of several diagnostic agents on the process of adipocyte differentiation of these two cells. No differences were found between these two lines of cells in the susceptibility to the antiadipogenic action of 12-0-tetradecanoylphorbol-13-acetate (TPA), or to TNFalpha, indicating that the basic biochemical components engaged in the antiadipogenic actions of these agents in these two cell lines are similar. In contrast, F442A cells were found to be more resistant to the antiadipogenic action of EGF or TGFbeta than L1 cells which were tested side by side. Based on the knowledge that TNFalpha preferentially affects C/EBPalpha and that TGFbeta specifically controls C/EBPbeta and delta in their antiadipogenic action, we hypothesized that the major cause for the differential response of these two similar cell lines could be the insensitivity of C/EBPbeta and/or delta of F442A cells to the action of TCDD. We could obtain supporting data for this hypothesis, showing that in F442A cells, the level of C/EBPbeta is already high even before the addition of adipocyte differentiation factors and that TCDD did not cause any significant changes in the titer of C/EBPbeta.     

Cord blood CD4CD25 regulatory T cells fail to inhibit cord blood NK cell functions due to insufficient production and expression of TGF-beta1

Although CD4⁺CD25⁺ Treg (Treg) cells are known to modulate NK cell functions, the modulation mechanism of these cells in cord blood has not been fully clarified. The purpose of this study was to clarify the mechanism whereby cord blood Treg cells modulate cord NK cells. By performing various cultures of purified NK cells with or without autologous Treg cells, diminished inhibitory effects of cord Treg cells towards cord NK cell functions, including activation, cytokine production, and cytotoxicity, were observed. We also observed lower secretion of sTGF-beta1 and lower expression of mTGF-beta1 by cord Treg cells than by adult Treg cells. These data revealed the capability of adult Treg cells to suppress rhIL-2-stimulated NK cell function by TGF-beta1, both membrane-bound and soluble types. The reduced inhibitory capabilities of cord Treg cells compared with adult Treg cells is thought to be due to insufficient expression of TGF-beta1.