Treg Cell Phenotype Research Articles

Expression of T-plastin, FoxP3 and other tumor-associated markers by leukemic T-cells of cutaneous T-cell lymphoma

Peripheral blood cells from 28 patients with leukemic cutaneous T-cell lymphoma including 25 patients with Sézary syndrome were evaluated for expression of regulatory T-cell-associated markers (FoxP3, CD25, CTLA-4, neurophilin-1), T-cell activation markers (CD28 and its ligands B7.1 and B7.2) and NK cell-associated markers (NKG2D and its ligands Mic-A and Mic-B) using real-time quantitative polymerase chain reaction. T-plastin served as a positive genetic marker, and its expression correlated to blood tumor burden. More than 90% of samples had transcripts for CD28 and Mic-B, but less than 30% of samples expressed FoxP3, CTLA-4 and CD25. Expression of Mic-B by neoplastic cells could provide another mechanism to inhibit anti-tumor immune responses. FoxP3 expression correlated with a poor prognosis. Although the underlying mechanisms accounting for this correlation remain unclear, the expression of the Foxp3 and CTLA-4 regulatory elements indicates that a subset of leukemic cases displays a regulatory T-cell phenotype.

Human plasmacytoid dendritic cells from patients with chronic hepatitis B virus infection induce the generation of a higher proportion of CD4+ and CD25+ regulatory T cells compared with healthy patients

This study was undertaken to investigate whether plasmacytoid dendritic cells (PDC) are involved in the generation of a higher proportion of CD4(+) and CD25(+) regulatory T (Treg) cells in chronic hepatitis B virus (HBV) infection compared with healthy patients. The amount, phenotype, and function of Treg cells in CD4(+) T cells primed by PDC from 46 chronic HBV patients, 25 healthy controls, and 10 individuals with resolved HBV infection were studied by flow cytometry, reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and proliferation assay. CD4(+) T cells primed by PDC from chronic HBV patients were more effective than CD4(+) T cells primed by PDC from the healthy controls and the resolved HBV patients in suppressing the hepatitis B core antigen-specific proliferation and the interferon production. The interleukin-10 and transforming growth factor-beta1 could also be detected in the supernatants of the PDC-primed CD4(+) T cells. A higher percentage of Treg cells, defined as CD4,CD25, CD45RO, and cytotoxic T-lymphocyte-associated antigen 4-positive cells, was detected within the population of CD4(+) T cells primed by PDC from chronic HBV patients compared with the healthy controls and individuals with resolved HBV infection. Accordingly, CD25(+) Treg cells from PDC-primed CD4(+) T cells displayed a high Fox P3 messenger RNA level. Depleting CD4(+) and CD25(+) Treg cells from CD4(+) T cells primed by PDC from the chronic HBV patients, the healthy volunteers, and the resolved HBV patients made PDC-primed CD4(+) T lose the capability in suppressing HBV-specific T cells. PDC from the patients with chronic HBV infection induced the generation of a higher proportion of CD4(+) and CD25(+) Treg cells compared with the healthy patients.

Th-17 Lymphocytes, Not Regulatory T Cells, Mediate Immune Regulation of Bone Marrow Infiltrating Lymphocytes and Induce Osteoclast Activation in Multiple Myeloma.

The bone marrow of myeloma patients is a reservoir of marrow infiltrating lymphocytes (MILs) that upon activation with anti-CD3 and anti-CD28 antibodies show significant tumor specificity against both mature myeloma plasma cells and their clonotypic precursors, whereas activated peripheral blood lymphocytes (PBLs) fail to demonstrate any tumor specificity. Here we examine the major differences between MILs and PBLs of both myeloma patients and normal donors. In myeloma, CD4+/CD25+ MILs lack a regulatory T-cell (Treg) phenotype as shown by the absence of FoxP3 expression and the inability to suppress tumor-specific proliferation of effector T cells. In contrast, the CD4+/CD25+ PBLs express FoxP3 and suppress tumor specific proliferation. Analysis of MILs and PBLs from normal individuals reveals the exact opposite (more Tregs in the marrow than blood). Considering the abundance of plasma cell-derived IL-6 in myeloma marrow, we hypothesize that IL-6 skewing of MILs towards the effector Th17 phenotype could explain the reciprocal paucity of Tregs in myeloma and relative abundance in normal bone marrows. Intracellular staining shows increased expression of IL-17 in myeloma CD4 MILs (2.7%) as compared to either myeloma PBLs (0.02%) or normal MILs (0.7%). The Th17 cytokine secretion pattern also appears more pronounced in myeloma-derived MILs as compared to either myeloma PBLs or normal MILs respectively (see table). Th17 T cells have recently been implicated in osteoclastogenesis of rheumatoid arthritis. In fact, bone marrow serum IL-17 levels correlate with the degree of lytic bone disease. In experiments utilizing M-CSF and RANK-L as the osteoclast differentiation medium, we show that the addition of IL-17 to the culture medium increases the number of mature osteoclasts by 2.3 fold whereas γIFN reduces mature osteoclast numbers by 80%. These findings explain the profound differences seen in MILs of normal and myeloma patients and implicate Th17 MILs in the generation of lytic bone disease. Activation of myeloma MILs with anti-CD3/CD28 skews the population from Th17 to a γIFN-producing Th1 phenotype. As such, targeting Th17 T cells or utilizing adoptive T cell therapy with activated, γIFN-producing MILs may represent a novel therapeutic mechanism to target osteoclastogenesis and reduce bone disease in myeloma.Th-17 Cytokine ProfileN=56MM MILsMM PBLsNL MILsIL-6 (pg/ml)303.76.5IL-23 (pg/ml)24619.635.9IL-17 (pg/ml)12.20.45.1

In vitro expansion of CD4 +CD25 highFOXP3 +CD127 low/− regulatory T cells from peripheral blood lymphocytes of healthy Mycobacterium tuberculosis-infected humans

CD4 +CD25 highFOXP3 + regulatory T (Treg) cells have recently been found at elevated levels in the peripheral blood of tuberculosis patients, compared to Mycobacterium tuberculosis latently infected (LTBI) healthy individuals and non-infected controls. Here, we show that CD4 +CD25 highFOXP3 + T lymphocytes can be expanded in vitro from peripheral blood mononuclear cells (PBMC) of LTBI individuals, but not of uninfected controls by incubating them with BCG in the presence of TGF-β. These expanded cells from the PBMC of LTBI subjects expressed CTLA-4, GITR and OX-40, but were CD127 low/− and have therefore the phenotype of Treg cells. In addition, they inhibited in a dose-dependant manner the proliferation of freshly isolated mononuclear cells in response to polyclonal stimulation, indicating that they are functional Treg lymphocytes. In contrast, incubation of the PBMC with BCG alone preferentially induced activated CD4 + T cells, expressing CD25 and/or CD69 and secreting IFN-γ. These results show that CD4 +CD25 highFOXP3 + Treg cells can be expanded or induced in the peripheral blood of LTBI individuals in conditions known to predispose to progression towards active tuberculosis and may therefore play an important role in the pathogenesis of the disease.

Flow cytometry-based methods for studying signaling in human CD4 +CD25 +FOXP3 + T regulatory cells

T regulatory (Treg) cells have a fundamental role in the establishment and maintenance of peripheral tolerance. It is well established that Treg cells have a phenotype and function that is distinct from conventional T effector cells, although how these two T cell subsets differ in terms of molecular signaling cascades remains largely unknown. Analysis of signaling events in Treg cells using classical biochemistry has been hampered due to difficulties in isolating homogeneous populations and limited cell numbers. In order to overcome these challenges, we defined the optimal conditions for culture, in vitro expansion, and stimulation of human CD4 +CD25 + Treg and T effector cells to study intracellular signaling events by flow cytometry. In order to avoid the pitfalls associated with cell isolation based on CD25 expression, we developed methodology to analyze subpopulations of FOXP3 positive and negative cells from ex vivo CD4 + T cells. In addition to examination of ex vivo cells, we optimized expansion conditions for analysis of signaling in Treg and T effector cell lines. Using these methods, we found that human FOXP3 + Treg cells displayed a greater capacity to phosphorylate the extracellular regulated kinase (ERK) compared to T effector cells, upon TCR-mediated activation. In contrast, FOXP3 + Treg cells showed a significantly diminished capacity to phosphorylate AKT. This methodology provides a foundation for future investigation into the molecular events that regulate the phenotype and function of Treg cells, and may ultimately lead to the identification of Treg-cell specific therapeutic targets.

Modeling the proteome of a Marek's disease transformed cell line: a natural animal model for CD30 overexpressing lymphomas

Marek's disease (MD) in the chicken, caused by the highly infectious MD alpha-herpesvirus (MDV), is both commercially important and a unique, naturally occurring model for human T-cell lymphomas overexpressing the Hodgkin's disease antigen, CD30. Here, we used proteomics as a basis for modeling the molecular functions and biological processes involved in MDV-induced lymphomagenesis. Proteins were extracted from an MDV-transformed cell line and were then identified using 2-D LC-ESI-MS/MS. From the resulting 3870 cellular and 21 MDV proteins we confirm the existence of 3150 "predicted" and 12 "hypothetical" chicken proteins. The UA-01 proteome is proliferative, differentiated, angiogenic, pro-metastatic and pro-immune-escape but anti-programmed cell death, -anergy, -quiescence and -senescence and is consistent with a cancer phenotype. In particular, the pro-metastatic integrin signaling pathway and the ERK/MAPK signaling pathways were the two predominant signaling pathways represented. The cytokines, cytokine receptors, and their related proteins suggest that UA-01 has a regulatory T-cell phenotype.

CD4+CD25high Regulatory T Cells Are Markedly Decreased in Blood of Patients with Pemphigus Vulgaris

Background: It remains to be determined whether pemphigus vulgaris (PV), an autoimmune blistering disease, has a reduction and/or dysfunction of CD4⁺CD25^high regulatory T (Treg) cells. Objectives: To evaluate the frequency and phenotypes of Treg cells in blood of patients with PV. Methods: Peripheral blood mononuclear cells were prepared from PV patients as well as normal and disease control volunteers, and the frequency and phenotypes of Treg cells were determined by flow cytometry. CD4⁺CD25⁺ and CD4⁺CD25^– T cells isolated from peripheral blood mononuclear cells of PV patients and normal controls were subjected to real-time semiquantitative RT-PCR for the expression of Foxp3 gene. Results: The proportion of Treg cells in all PV patients was severely reduced, approximately ten times less than controls. These observations were further confirmed by both diminished gene and protein expression of Foxp3 in the CD4⁺CD25⁺ T cell population in PV patients. Conclusions: Numerical impairment of Treg cells may be involved in the pathogenesis of PV.

Regulatory T‐cell phenotype in association with large cell transformation of mycosis fungoides

Tumor cells of primary cutaneous T-cell lymphomas are able to adopt a regulatory T-cell phenotype in vitro. The significance of this finding in vivo is matter of debate. We stained five cases with transformed mycosis fungoides (MF) with an antibody against FOXP3, which is a sensitive and specific marker for the regulatory T-cell phenotype. Transformed T cells in four of five patients with MF stained positive for FOXP3. One patient who showed no CD30 expression of large transformed T cells was also negative for FOXP3. Comparison of plaques and tumors in one patient showed that FOXP3 and CD30 expression was exclusively observed in large transformed tumor cells whereas malignant T cells without large cell transformation were negative. Transformation of MF to high grade lymphoma may be associated with the adoption of a regulatory T-cell phenotype. FOXP3 expression may contribute to aggressive behavior of MF after large cell transformation via immune escape mechanism. The significance of this observation is limited by the low case number in this study.

Short-Term Treatment With Anti-CD3 Antibody Reduces the Development and Progression of Atherosclerosis in Mice

Atherosclerosis is a chronic inflammatory disease of the large arteries that is the primary cause of heart disease and stroke. Anti-CD3-specific antibodies suppress immune responses by antigenic modulation of the CD3 antibody/T-cell receptor complex. Their unique capacity to restore self-tolerance in a mouse model of diabetes and, importantly, in patients with recent-onset type 1 diabetes involves transforming growth factor-beta-dependent mechanisms via expansion and/or activation of regulatory T cells. We hypothesized that treatment with anti-CD3-specific antibodies might inhibit atherosclerosis development and progression in mice. Low-density lipoprotein receptor-deficient mice were fed a high-cholesterol diet for 13 or 24 weeks. Anti-CD3 antibody was administered on 5 consecutive days beginning 1 week before or 13 weeks after the high-cholesterol diet was initiated, respectively. Control mice were injected in parallel with phosphate-buffered saline. Anti-CD3 antibody therapy reduced plaque development when administered before a high-cholesterol diet and markedly decreased lesion progression in mice with already established atherosclerosis. We found increased production of the antiinflammatory cytokine transforming growth factor-beta in concanavalin A-stimulated lymph node cells and enhanced expression of the regulatory T-cell marker Foxp3 in spleens of anti-CD3 antibody-treated mice. A higher percentage of apoptotic cells within the plaques of anti-CD3 antibody-treated mice was also observed. Altered disease progression, combined with the emergence of this particular cytokine pattern, indicates that short-term treatment with an anti-CD3 antibody induces a regulatory T-cell phenotype that restores self-tolerance in a mouse model of atherosclerosis.

Nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) oncoprotein induces the T regulatory cell phenotype by activating STAT3

The mechanisms of malignant cell transformation mediated by the oncogenic, chimeric nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) tyrosine kinase remain only partially understood. Here we report that the NPM/ALK-carrying T cell lymphoma (ALK+TCL) cells secrete IL-10 and TGF-beta and express FoxP3, indicating their T regulatory (Treg) cell phenotype. The secreted IL-10 suppresses proliferation of normal immune, CD3/CD28-stimulated peripheral blood mononuclear cells and enhances viability of the ALK+TCL cells. The Treg phenotype of the affected cells is strictly dependent on NPM/ALK expression and function as demonstrated by transfection of the kinase into BaF3 cells and inhibition of its enzymatic activity and expression in ALK+TCL cells. NPM/ALK, in turn, induces the phenotype through activation of its key signal transmitter, signal transducer and activator of transcription 3 (STAT3). These findings identify a mechanism of NPM/ALK-mediated oncogenesis based on induction of the Treg phenotype of the transformed CD4(+) T cells. These results also provide an additional rationale to therapeutically target the chimeric kinase and/or STAT3 in ALK+TCL.

Expression of regulatory T-lymphocyte phenotype in human fetal hemopoietic and lymphoid cell culture

Cells with regulatory T-cell phenotype (Treg, CD4+CD25(hi)) were not detected in human fetal thymus, liver, bone marrow, spleen, and among blood mononuclears of 14-28-week gestation. The cells of the majority of these fetal thymuses express Treg specific marker (FOXP3 transcription marker) gene. Culturing of fetal liver and bone marrow cells on a monolayer of thymic epithelial cells induced expression of FOXP3 gene, but induction of CD4+CD25+ membrane phenotype was detected in only 1 of 8 studied cultures (in liver and bone marrow cells). Induction of Treg differentiation is to a greater extent determined by the characteristics of hemopoietic organ cells than of thymic epithelial cells.

Foxp3 Expressing CD4 + CD25 + and CD8 +CD28 − T Regulatory Cells in the Peripheral Blood of Patients with Lung Cancer and Pleural Mesothelioma

The role of T regulatory (Treg) cells in human cancer has not yet been clarified. We assessed the presence and function of CD4 + and CD8 + Treg cell subsets in the peripheral blood of patients with lung cancer (LC) and pleural mesothelioma (PM). We found a low but significant increase in the number of CD4 + T cells with phenotype and functional features of Treg cells in LC patients compared to normal healthy controls (NHC). Furthermore, total CD4 + T cells from LC patients proliferated less than cells from controls, suggesting that the increase in the CD4 + Treg cell pool has functional importance. LC patients also showed an expansion of the CD8 +CD28 − T cell subset and these cells expressed Foxp3 mRNA, as recently observed in alloantigen-specific CD8 +CD28 − T suppressor cells. No variation of peripheral Treg cell subsets was found in patients with PM, a disease with a predominantly localized nature. However, the lack of correlation between cancer stage and the number or the function of peripheral Treg cells in LC patients refuted the hypothesis that these cells are involved in tumor spreading. A possible involvement of the peripheral Treg cell pool in cancer development and/or in inducing systemic immunosuppression in LC patients can be hypothesized.

CD4+CD25+ regulatory T cells: II. Origin, disease models and clinical aspects

Autoimmune diseases afflict approximately 5% of the population and reflect a failure in the immune system to discriminate between self and non-self resulting in the breakdown of self-tolerance. Regulatory CD4+CD25+ T cells (Treg cells) have been shown to play an important role in the maintenance of immune homeostasis and self-tolerance by counteracting the development and effector functions of potentially autoreactive T cells. We have in the previous APMIS review described the phenotype and physiology of Treg cells. The present overview deals with the thymic origin of Treg cells and their role in disease models such as autoimmune gastritis and inflammatory bowel disease. Finally, we will consider some aspects of the therapeutic potential of Treg cells.

Overexpression of the Notch ligand, Jagged-1, induces alloantigen-specific human regulatory T cells

AbstractGraft-versus-host disease (GVHD) represents one of the major complications of allogeneic hematopoietic stem cell transplantation. Techniques to prevent GVHD have included ex vivo T-cell depletion of the graft or prolonged in vivo immunosuppression. Both reduce the frequency and severity of GVHD but also reduce T-cell-mediated graft-versus-malignancy effect, and increase the risk of infection. A major goal in transplantation is to prevent alloreactivity while preserving activity against tumors and infectious agents. We have used activation of the Notch pathway to try to generate T cells able to specifically regulate alloantigen responses. We used allogeneic Epstein-Barr virus lymphoblastoid B cells (EBV-LCLs) as stimulator cells. Such LCLs are excellent (allo) antigen-presenting cells and can be obtained in large numbers even from donors who have received extensive chemo/radiotherapy. We overexpressed a Notch ligand, Jagged-1, in these cells by adenoviral vector transduction. Stimulation of CD45RA+ naive T cells by Jagged-1 EBV-LCL reduces production of interferon-γ, interleukin-2, and interleukin-5, but up-regulates transforming growth factor-β1 synthesis, consistent with induction of a regulatory T-cell phenotype. Transfer of these T cells to fresh lymphocyte cultures inhibits proliferative and cytotoxic immune responses to the priming alloantigens while sparing responses to third-party stimulator cells. Notch activation in the presence of alloantigen-presenting cells may therefore be a means of inducing specific regulatory T cells while preserving other T-cell functionality. (Blood. 2003;102:3815-3821)

Lymphocyte populations in human lymph nodes. Alterations in CD4+ CD25+ T regulatory cell phenotype and T-cell receptor Vbeta repertoire.

Here we provide a description of lymphocyte populations in human lymph nodes (LN) with a special emphasis on the CD4+ lymphocyte population constitutively expressing CD25 at a high level and endowed with immunoregulatory properties [T regulatory (Treg) cells]. Lymph nodes were analysed by multicolour flow cytometry in parallel with correspondent peripheral blood (PB). Immunomagnetically purified Treg cells were tested for anergy and suppressive activity in a CD3/T-cell receptor (TCR)-driven proliferation assay. Compared to PB, there was a reduced T/B lymphocyte ratio in LN. Both LN and PB contained a similar proportion of CD4+ lymphocytes but, conversely, CD8+ lymphocytes were less represented in PB, with a consequent increase in the ratio of CD4+/CD8+ natural killer cells were <2% (PB range 6-22%). No significant differences existed in the frequency of the other lymphocyte subpopulations examined (naïve-type CD4+ and CD8+ lymphocytes, activated B and CD4+ lymphocytes, and effector-type CD8+ lymphocytes). LN and PB contained similar percentages of CD4+ lymphocytes constitutively expressing intermediate or high levels of CD25. CD4+ CD25++ cells constitutively coexpressed high levels of CD152 and were therefore identified as Treg cells. Treg cells in LN and PB differed in terms of CD45RB, HLA-DR, CD45RO, and CD62L expression. Also the TCRVbeta repertoire diverged between Treg cells from LN and PB. Similar to Treg cells from PB, Treg cells from LN were anergic and efficiently inhibited other CD4+ and CD8+ lymphocyte proliferation. This study extends the information on the diversities in lymphocyte composition between human LN and PB, and reports for the first time a description of the phenotypic and functional characteristics of Treg cells in human LN, highlighting the importance of the LN microenvironment in shaping the surface phenotype of Treg cells.

Heat shock proteins, inflammation, and cardiovascular disease.

Atherosclerosis and its associated complications, such as coronary artery disease, peripheral vascular disease, and stroke, are the leading causes of morbidity and mortality in all racial groups of most westernized societies. A better understanding of the events that lead to the induction and progression of atherosclerosis and the development of strategies that control these processes would have a significant impact on human health. Atherosclerosis is a chronic inflammatory condition that usually begins at an early age in the absence of lipid accumulation, with fatty streaks composed of lipid-laden macrophages (foam cells) developing at later stages. T lymphocytes, predominantly of the CD4+ phenotype, are associated with the intima layer of the vessel. The inflammatory process at atherogenic sites leads to the production of cytokines and other inflammatory mediators, resulting in cell migration, proliferation, extracellular matrix production, and plaque development.1–3⇓⇓ Monocytes and macrophages appear intimately involved with the development of atherosclerosis; mice with the osteopetrotic ( op ) mutation in the macrophage colony-stimulating factor (M-CSF) gene, which results in a complete absence of M-CSF in the serum and tissues and a marked reduction in the number of circulating monocytes, exhibit significantly less atherosclerosis than control littermates when bred into an apolipoprotein (apo) E–deficient background,.4 The precise role of T and B cells in atherosclerosis remains unclear. Although activated T cells are a dominant feature of atherosclerotic lesions,5,6⇓ studies demonstrating that atherosclerosis can be induced by hypercholesterolemia in mice deficient in T and B cells suggest that these cells are not essential.7–9⇓⇓ However, the cholesterol levels in these studies far exceeded those present in human subjects without any genetic defect, and the findings should therefore be interpreted with caution. Indeed, other evidence indicates that T cells may attenuate atherogenesis by some means, because the elimination of …