IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo

IL-2 plays a critical role in the maintenance of CD4+CD25+ FOXP3(+) regulatory T cells (Tregs) in vivo. We examined the effects of IL-2 signaling in human Tregs. In vitro, IL-2 selectively up-regulated the expression of FOXP3 in purified CD4+CD25+ T cells but not in CD4+CD25- cells. This regulation involved the binding of STAT3 and STAT5 proteins to a highly conserved STAT-binding site located in the first intron of the FOXP3 gene. We also examined the effects of low-dose IL-2 treatment in 12 patients with metastatic cancer and 9 patients with chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation. Overall, IL-2 treatment resulted in a 1.9 median fold increase in the frequency of CD4+CD25+ cells in peripheral blood as well as a 9.7 median fold increase in FOXP3 expression in CD3+ T cells. CD56+CD3- natural killer (NK) cells also expanded during IL-2 therapy but did not express FOXP3. In vitro treatment of NK cells with 5-aza-2'-deoxycytidine restored the IL-2 signaling pathway leading to FOXP3 expression, suggesting that this gene was constitutively repressed by DNA methylation in these cells. Our findings support the clinical evaluation of low-dose IL-2 to selectively modulate CD4+CD25+ Tregs and increase expression of FOXP3 in vivo.     

Naive regulatory T cells: a novel subpopulation defined by resistance toward CD95L-mediated cell death

Most CD4(+)CD25(hi)FOXP3(+) regulatory T cells (T(regs)) from adult peripheral blood express high levels of CD45RO and CD95 and are prone to CD95L-mediated apoptosis in contrast to conventional T cells (T(convs)). However, a T(reg) subpopulation remained consistently apoptosis resistant. Gene microarray and 6-color flow cytometry analysis including FOXP3 revealed an increase in naive T-cell markers on the CD95L-resistant T(regs) compared with most T(regs). In contrast to T(regs) found in adult humans, most CD4(+)CD25(+)FOXP3(+) T cells found in cord blood are naive and exhibit low CD95 expression. Furthermore, most of these newborn T(regs) are not sensitive toward CD95L similar to naive T(regs) from adult individuals. After short stimulation with anti-CD3/CD28 monoclonal antibodies (mAbs), cord blood T(regs) strongly up-regulated CD95 and were sensitized toward CD95L. This functional change was paralleled by a rapid up-regulation of memory T-cell markers on cord blood T(regs) that are frequently found on adult memory T(regs). In summary, we show a clear functional difference between naive and memory T(regs) that could result in different survival rates of those 2 cell populations in vivo. This new observation could be crucial for the planning of therapeutic application of T(regs).     

Abnormal killer inhibitory receptor expression on natural killer cells in patients with Behçet's disease

Viral infection has been assigned some role in the pathogenesis of Behçet's disease (BD). Defects in natural killer (NK) cell repertoire may be involved in impaired antiviral immunity, leading to the development of BD. We studied killer inhibitory receptor (KIR) expression in 40 patients with BD. CD94 and CD158b expression of NK cells was normal in a great majority of BD patients. NKB1 expression was reduced in eight and increased in six. Twelve of these 14 patients (86%) had severe eye disease. Some had reduced NKB1 and enhanced CD158a expression simultaneously, or enhanced NKB1 and reduced CD158a simultaneously, suggesting a skewed NK cell repertoire in BD. Collectively, KIR expression was abnormal in the BD patients with severe eye disease. This may result from genetic predisposition, or certain viruses may affect the KIR repertoire formation in BD patients. Abnormal KIR expression of NK cells may be associated with the development of BD.     

Dendritic cells are specialized accessory cells along with TGF- for the differentiation of Foxp3+ CD4+ regulatory T cells from peripheral Foxp3 precursors

Foxp3(+)CD25(+)CD4(+) regulatory T cells are produced in the thymus (natural T regs) but can also differentiate from peripheral Foxp3(-)CD4(+) precursors (induced or adaptive T regs). We assessed antigen presenting cell (APC) requirements for the latter differentiation. With added transforming growth factor (TGF)-beta, both immature and mature populations of dendritic cells (DCs) induced antigen-specific Foxp3(+) T regs from Foxp3(-) precursors. Using endogenous TGF-beta, DCs from gut-associated mesenteric lymph nodes were capable of differentiating Foxp3(+)T regs. Spleen DCs were 100-fold more potent than DC-depleted APCs for the induction of T regs and required 10-fold lower doses of peptide antigen. Interleukin-2 (IL-2) was essential, but could be provided endogenously by T cells stimulated by DCs, but not other APCs. The required IL-2 was induced by DCs that expressed CD80/CD86 costimulatory molecules. The DC-induced Foxp3(+)T regs divided up to 6 times in 6 days and were comprised of CD62L and CD103 positive and negative forms. The induced Foxp3(+)T regs exerted suppression in vitro and blocked tumor immunity in vivo. These results indicate that DCs are specialized to differentiate functional peripheral Foxp3(+)T regs and help set the stage to use DCs to actively suppress the immune response in an antigen-specific manner.     

Mucosal but not peripheral FOXP3+ regulatory T cells are highly increased in untreated HIV infection and normalize after suppressive HAART

Recent evidence indicates that regulatory T cells (T(regs)) play an important role in HIV infection. However, although the gastrointestinal mucosa is a key compartment in HIV disease, no data on mucosal T(regs) in HIV infection are available. In this study, we compared the frequency of T(regs) in duodenal mucosa and peripheral blood (PB) of 13 treatment-naive and 13 suppressively treated HIV-infected patients with that of 6 patients with norovirus infection and 12 healthy controls. T(regs) were quantified by immunohistochemistry (CD3/FOXP3) and further characterized (CD25, CTLA-4, GITR) by immunohistochemistry, immunofluorescence, and fluorescence-activated cell sorting (FACS). Both the frequency and the absolute count of mucosal T(regs) were highly increased in untreated HIV patients but were normal in treated HIV patients. In contrast, in peripheral blood of HIV patients, the absolute number of T(regs) was not increased, and their frequency was only slightly elevated. In norovirus infection, frequency of mucosal T(regs) in the CD4+ T-cell subset was not elevated. The high increase in count and frequency of mucosal T(regs) seems to be a characteristic feature of untreated HIV infection, suggesting a significant contribution of T(regs) to the pathogenesis of HIV disease. Their role may be 2-edged: attenuating HIV-induced immune hyperactivation while suppressing the immune response to HIV and mucosal pathogens.     

Induction and role of regulatory CD4+CD25+ T cells in tolerance to the transgene product following hepatic in vivo gene transfer

Gene replacement therapy is complicated by the risk of an immune response against the therapeutic transgene product, which in part is determined by the route of vector administration. Our previous studies demonstrated induction of immune tolerance to coagulation factor IX (FIX) by hepatic adeno-associated viral (AAV) gene transfer. Using a regulatory T-cell (T(reg))-deficient model (Rag-2(-/-) mice transgenic for ovalbumin-specific T-cell receptor DO11.10), we provide first definitive evidence for induction of transgene product-specific CD4(+)CD25(+) T(regs) by in vivo gene transfer. Hepatic gene transfer-induced T(regs) express FoxP3, GITR, and CTLA4, and suppress CD4(+)CD25(-) T cells. T(regs) are detected as early as 2 weeks after gene transfer, and increase in frequency in thymus and secondary lymphoid organs during the following 2 months. Similarly, adoptive lymphocyte transfers from mice tolerized to human FIX by hepatic AAV gene transfer indicate induction of CD4(+)CD25(+)GITR(+) that suppresses antibody formation to FIX. Moreover, in vivo depletion of CD4(+)CD25(+) T(regs) leads to antibody formation to the FIX transgene product after hepatic gene transfer, which strongly suggests that these regulatory cells are required for tolerance induction. Our study reveals a crucial role of CD4(+)CD25(+) T(regs) in preventing immune responses to the transgene product in gene transfer.     

IL-2-independent generation of FOXP3(+)CD4(+)CD8(+)CD25(+) cytotoxic regulatory T cell lines from human umbilical cord blood

OBJECTIVE: Since the existence of mouse naturally occurring CD4(+)CD25(+) T regulatory (Treg) cells was demonstrated, a variety of human Treg subsets have been identified as distinct T cell populations. Here we show the establishment of novel Treg cell lines possessing unique characteristics. METHODS: Novel Treg cell lines, designated HOZOT, were generated by coculturing human umbilical cord blood cells with mouse stromal cell lines in the absence of exogenous IL-2 or other cytokines. HOZOT were characterized and compared with CD4(+)CD25(+) Treg cells in terms of the CD phenotype, FOXP3 expression, suppressor activity against allogeneic MLR, anergy property, and IL-10 production. RESULTS: HOZOT were generated and expanded as normal lymphoblastoid cells with cytotoxic activity against the cocultured stromal cells. HOZOT consisted of three subpopulations as defined by phenotype: CD4(+)CD8(+), CD4(+)CD8(dim), and CD4(-)CD8(+). All three subpopulations showed both suppressor and cytotoxic activities. While HOZOT's expression of FOXP3, CD25, GITR, and cytoplasmic CTLA-4 implied a similarity to naturally occurring CD4(+)CD25(+) Treg cells, these two Treg cells differed in IL-2 responsiveness and IL-10 production. CONCLUSIONS: Our studies introduce a new method of generating Treg cells in an IL-2-independent manner and highlight a unique Treg cell type with cytotoxic activity and a phenotype of FOXP3(+)CD4(+)CD8(+)CD25(+).     

CD147 (Basigin/Emmprin) identifies FoxP3+CD45RO+CTLA4+-activated human regulatory T cells

Human CD4(+)FoxP3(+) T cells are functionally and phenotypically heterogeneous providing plasticity to immune activation and regulation. To better understand the functional dynamics within this subset, we first used a combined strategy of subcellular fractionation and proteomics to describe differences at the protein level between highly purified human CD4(+)CD25(+) and CD4(+)CD25(-) T-cell populations. This identified a set of membrane proteins highly expressed on the cell surface of human regulatory T cells (Tregs), including CD71, CD95, CD147, and CD148. CD147 (Basigin or Emmprin) divided CD4(+)CD25(+) cells into distinct subsets. Furthermore, CD147, CD25, FoxP3, and in particular CTLA-4 expression correlated. Phenotypical and functional analyses suggested that CD147 marks the switch between resting (CD45RA(+)) and activated (CD45RO(+)) subsets within the FoxP3(+) T-cell population. Sorting of regulatory T cells into CD147(-) and CD147(+) populations demonstrated that CD147 identifies an activated and highly suppressive CD45RO(+) Treg subset. When analyzing CD4(+) T cells for their cytokine producing potential, CD147 levels grouped the FoxP3(+) subset into 3 categories with different ability to produce IL-2, TNF-α, IFN-γ, and IL-17. Together, this suggests that CD147 is a direct marker for activated Tregs within the CD4(+)FoxP3(+) subset and may provide means to manipulate cells important for immune homeostasis.     

Expansion of FOXP3high regulatory T cells by human dendritic cells (DCs) in vitro and after injection of cytokine-matured DCs in myeloma patients

CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg's) play an important role in the maintenance of immune tolerance. The mechanisms controlling the induction and maintenance of Treg's in humans need to be defined. We find that human myeloid dendritic cells (DCs) are superior to other antigen presenting cells for the maintenance of FOXP3(+) Treg's in culture. Coculture of DCs with autologous T cells leads to an increase in both the number of Treg's, as well as the expression of FOXP3 protein per cell both in healthy donors and myeloma patients. DC-mediated expansion of FOXP3(high) Treg's is enhanced by endogenous but not exogenous interleukin-2 (IL-2), and DC-T-cell contact, including the CD80/CD86 membrane costimulatory molecules. DCs also stimulate the formation of Treg's from CD25(-) T cells. The efficacy of induction of Treg's by DCs depends on the nature of the DC maturation stimulus, with inflammatory cytokine-treated DCs (Cyt-DCs) being the most effective Treg inducers. DC-induced Treg's from both healthy donors and patients with myeloma are functional and effectively suppress T-cell responses. A single injection of cytokine-matured DCs led to rapid enhancement of FOXP3(+) Treg's in vivo in 3 of 3 myeloma patients. These data reveal a role for DCs in increasing the number of functional FOXP3(high) Treg's in humans.     

Expression of KIR and C-type lectin receptors in Behcet's disease

OBJECTIVE: Beh?et's disease (BD) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. Genetic susceptibility associated with HLA-B*51 and B*2702 has been implicated in its pathogenesis. Considering the recently defined regulatory mechanisms of NK cells through HLA class I binding receptors, we hypothesized that interactions of NK and T cells through the NK receptors may be important in the pathogenesis of BD. METHODS: The impact of different expression patterns of HLA-recognizing receptors on NK or T cells was analysed in 51 patients with BD and 32 healthy controls. We used flow cytometry to investigate the expression of KIR3DL1 from the polymorphic killer immunoglobulin-like receptor (KIR) family, which binds a shared HLA-Bw4 motif on HLA-B51 and *2702 alleles, and CD94 from the conserved C-type lectin receptor family, which binds HLA-E. Thirty-three of the BD patients and 19 of the controls carried the same HLA-Bw4 motif. RESULTS: CD3(+) T cells were increased in patients with BD compared with controls (81 vs 75%, P = 0.001), whereas the NK cells did not show any difference between the two groups. Increased expression of CD94 in BD was observed on CD16(+)CD56(+) cells (66 vs 57, P = 0.04) and on CD3(+) (7.7 vs 4.0, P < 0.001) and CD3(+)CD56(+) (44 vs 35, P = 0.02) T cells. KIR3DL1 expression on the NK and T cells was not statistically different between the two groups. No effect of HLA-Bw4 motif was observed on the expression of CD94 and KIR3DL1 in both the patients and the controls. CONCLUSION: The absence of a correlation between KIR3DL1 expression and HLA-Bw4 motif confirms previous work reporting that the expression of these molecules is regulated separately. Increased expression of CD94 may suggest that NK receptors play a pathogenic or regulatory role in BD.     

Calcineurin-dependent negative regulation of CD94/NKG2A expression on naive CD8+ T cells

Immune responses lead to expression of immunoregulatory molecules on T cells, including natural killer (NK) receptors, such as CD94/NKG2A on CD8(+) T cells; these receptors restrain CD8(+) responses, thereby preventing T-cell exhaustion in chronic infections and limiting immunopathology. Here, we examined the requirements for inducing CD94/NKG2A on T cells responding to antigen. In vitro, moderate induction of CD94/NKG2A expression occurred after exposure of naive CD8(+) (but not CD4(+)) cells to CD3 ligation or specific peptide. Surprisingly, expression was inhibited by CD28/B7 costimulation. Such inhibition applied only to CD94/NKG2A and not other NK receptors (NKG2D) and was mediated by IL-2. Inhibition by IL-2 occurred via a NFAT cell-independent component of the calcineurin pathway, and CD94/NKG2A induction was markedly enhanced in the presence of calcineurin blockers, such as FK506 or using calcineurin-deficient T cells, both in vitro and in vivo. In addition to CD28-dependent inhibition by IL-2, CD94/NKG2A expression was impaired by several other cytokines (IL-4, IL-23, and transforming growth factor-β) but enhanced by others (IL-6, IL-10, and IL-21). The complex interplay between these various stimuli may account for the variable expression of CD94/NKG2A during responses to different pathogens in vivo.     

BCR/ABL alters the function of NK cells and the acquisition of killer immunoglobulin-like receptors (KIRs)

Natural killer (NK) cells decrease in function during chronic myelogenous leukemia (CML) progression from chronic phase to blast crisis, and they can become BCR/ABL(+) late in the disease course. To study this altered function, NK92 cells were transduced with the BCR/ABL oncogene. In contrast to the parental cells, which died when deprived of interleukin 2 (IL-2), p210(+) NK92 cells proliferated and survived indefinitely in the absence of IL-2. BCR/ABL also decreased the natural cytotoxicity of NK92 cells against K562 targets, without affecting IL-2, interferon gamma (IFN-gamma), or tumor necrosis factor alpha (TNF-alpha) production. Although the ABL-specific tyrosine kinase inhibitor imatinib mesylate (STI-571) had no effect on parental NK92 cells, it markedly decreased the growth and survival of IL-2-independent p210(+) NK92 cells. In contrast to the parental cell line, serial analysis of p210(+) NK92 cells detected small populations that clonally expressed one or more killer immunoglobulin-like receptors (KIRs). Unlike the decreased natural cytotoxicity, the function of the activating CD158j receptor remained intact. Southern blotting and hybridization with an enhanced green fluorescence protein (eGFP) probe showed that KIR(-) and KIR(+) NK92 cells were all derived from the same clone, suggesting that KIR acquisition remains dynamic at the maturational stage represented by the NK92 cell line. When tested in primary CD56(+bright) NK cells, p210 induced partial IL-2-independent growth and increased KIR expression similar to findings in NK92 cells. This is the first study to show that BCR/ABL, well known for its effects on the myeloid lineage, can alter the function of lymphoid cells, which may be associated with the defect in innate immunity associated with CML progression.     

Increased population of CD4(+)CD25(high), regulatory T cells with their higher apoptotic and proliferating status in peripheral blood of acute myeloid leukemia patients

PURPOSE: Regulatory T cells (T-reg) that control harmful autoimmune T cells in the periphery may also suppress the immune response against cancer. In this study we investigated the possible involvement of CD4(+)CD25(high) T-reg in the immune impairment of patients with acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: The frequencies and phenotypes of CD4(+)CD25(high) T cells in the peripheral blood of AML patients were determined by flow cytometry. To assess the functional activity of CD4(+)CD25(high) T cells, CD4(+)CD25(high), and CD4(+)CD25(-) T cells were sorted from peripheral blood mononuclear cells with FACS Vantage. The immunoregulatory properties of CD4(+)CD25(high) and CD4(+)CD25(-) T cells were characterized by proliferation assays and cytokine production assays. In addition, the frequency of apoptotic and proliferating cells in CD4(+)CD25(high) T cells were respectively evaluated by 7AAD and ki67 binding cells using flow cytometry. RESULTS: Compared with healthy controls, AML patients had a higher proportion of CD4(+)CD25(high) T cells in peripheral blood. These cells were CD45-RA(-), CD69(-), CD45-RO(+), CD95(+), and intercellular CTLA-4(+), and secreted low levels of TNF-alpha and IL-10, but no IL-2, IL-4, IL-5, and IFN-gamma. They inhibited the proliferation and cytokine production (IL-2, IFN-gamma) of CD4(+)CD25(-) T cells, but improved IL-10 production under the co-culture of both subsets with stimulation, thus behaving as T-reg. Notably, CD4(+)CD25(high) T cells in AML patients presented significantly higher apoptosis and proliferation than that of healthy individuals. CONCLUSIONS: The frequency of CD4(+)CD25(high) T-reg in peripheral blood in AML patients is significantly higher when compared with healthy individuals, likely due to the increasing proliferation of CD4(+)CD25(high) T cells.     

FOXP3 expression accurately defines the population of intratumoral regulatory T cells that selectively accumulate in metastatic melanoma lesions

Regulatory T (T(reg)) cells are often found in human tumors; however, their functional characteristics have been difficult to evaluate due to low cell numbers and the inability to adequately distinguish between activated and T(reg) cell populations. Using a novel approach, we examined the intracellular cytokine production capacity of tumor-infiltrating T cells in the single-cell suspensions of enzymatically digested tumors to differentiate T(reg) cells from effector T cells. Similar to T(reg) cells in the peripheral blood of healthy individuals, tumor-infiltrating FOXP3(+)CD4 T cells, unlike FOXP3(-) T cells, were unable to produce IL-2 and IFN-gamma upon ex vivo stimulation, indicating that FOXP3 expression is a valid biological marker for human T(reg) cells even in the tumor microenvironment. Accordingly, we enumerated FOXP3(+)CD4 T(reg) cells in intratumoral and peritumoral sections of metastatic melanoma tumors and found a significant increase in proportion of FOXP3(+)CD4 T(reg) cells in the intratumoral compared with peritumoral areas. Moreover, their frequencies were 3- to 5-fold higher in tumors than in peripheral blood from the same patients or healthy donors, respectively. These findings demonstrate that the tumor-infiltrating CD4 T(reg) cell population is accurately depicted by FOXP3 expression, they selectively accumulate in tumors, and their frequency in peripheral blood does not properly reflect tumor microenvironment.     

CD4+,CD28- T cells in rheumatoid arthritis patients combine features of the innate and adaptive immune systems

OBJECTIVE: To determine whether CD4+,CD28- T cells, which are expanded in patients with rheumatoid arthritis (RA), express receptors that typically regulate the function of natural killer (NK) cells. METHODS: Expression of the NK cell surface molecules CD158, p70, CD94, CD161, and CD8alpha on T cell subsets was determined by multicolor flow cytometric analysis of peripheral blood mononuclear cells from 36 RA patients. Expression of CD161 on tissue-infiltrating CD4 T cells was determined by 2-color immunohistochemistry analysis of synovial tissue samples. RESULTS: Killer cell-inhibitory receptors (KIR) and killer cell-activating receptors (KAR) were exclusively expressed on CD4+,CD28- T cells, with the CD158b molecule being the most frequently detected isoform. A coordinated mechanism inducing KIR/KAR expression was suggested by similarities in the expression of CD158b on CD4 and CD8 T cells. CD4+,CD28- T cells were also positive for CD8-alphaalpha homodimers, another characteristic shared with NK cells. Of the C-type lectin NK cell receptors (NK receptors), CD94 was consistently absent, but CD161 was found on a CD4 T cell population that is significantly expanded in RA patients (P = 0.01). Involvement in disease of NK receptor-expressing CD4 T cells was suggested by the presence of CD4+,CD161+ T cells in follicular microstructures typical of rheumatoid synovitis. CONCLUSION: Patients with RA have an expanded and unusual subset of CD4 T cells that infiltrates the tissue lesions and is characterized by a deficiency of CD28, the expression of CD8-alphaalpha homodimers, and the expression of several types of HLA class I-recognizing NK receptors. CD4 T cells bearing NK receptors can bridge functions of the innate and adaptive immune systems, such as responsiveness to specific antigen, rapid release of interferon-gamma, cytotoxicity, independence from classic costimulatory pathways, and integration of multiple activating and inhibitory signals to control effector functions.     

The unexpected effect of cyclosporin A on CD56+CD16- and CD56+CD16+ natural killer cell subpopulations

Cyclosporin A (CSA) is commonly used to prevent graft-versus-host disease. The influence of CSA on T-cell function has been extensively investigated; however, the effect of CSA on natural killer (NK) cells is less understood. NK cells were cultured with IL-2 and IL-15 with and without CSA for 1 week. Compared with controls, CSA-treated cultures showed fewer CD56(+)CD16(+)KIR(+) NK cells and a reciprocal increase in CD56(+)CD16(-)KIR(-) cells. These changes were due mainly to a reduced proliferation of the CD56(dim) NK-cell subpopulation and a relative resistance of CD56(bright) NK cells to CSA. Following coculture with K562 targets, CSA-exposed NK cells differed from controls and lacked Ca(2+) oscillations, nuclear factor of activated T cells (NFAT) dephosphorylation, and NFAT nuclear translocation. NK cells cultured in CSA retained cytotoxicity against K562, Raji, and KIR ligand-expressing lymphoblastoid cells. NK cells cultured in CSA showed increases in NKp30 and reductions in NKp44 and NKG2D. Following IL-12 and IL-18 stimulation, CSA-treated NK cells showed more IFN-gamma-producing cells. Using in vitro NK-cell differentiation, progenitor cells gave rise to more CD56(+)KIR(-) NK cells in the presence of CSA than controls. Collectively, these studies show that CSA influences NK-cell function and phenotype, which may have important implications for graft-versus-leukemia effects.