Down-regulation of IL-7Ralpha expression in human T cells via DNA methylation

IL-7 is critical for the development and survival of T cells. Recently, we found two subsets of human CD8+ T cells expressing IL-7Ralpha(high) and IL-7Ralpha(low) with different cell survival responses to IL-7. Although these CD8+ T cell subsets have differential IL-7Ralpha gene expression, the mechanism for this is unknown. DNA methylation is an important gene regulatory mechanism and is associated with the inactivation of gene expression. Thus, we investigated a role for DNA methylation in differentially regulating IL-7Ralpha gene expression in human CD8+ T cells and Jurkat T cells. IL-7Ralpha(high)CD8+ T cells had decreased methylation in the IL-7Ralpha gene promoter compared with IL-7Ralpha(low)CD8+ T cells and Jurkat T cells with low levels of IL-7Ralpha. Treating Jurkat T cells with 5-aza-2'-deoxycytidine, which reduced DNA methylation, increased IL-7Ralpha expression. Plus, the unmethylated IL-7Ralpha gene promoter construct had higher levels of promoter activity than the methylated one as measured by a luciferase reporter assay. These findings suggest that DNA methylation is involved in regulating IL-7Ralpha expression in T cells via affecting IL-7Ralpha gene promoter activity, and that the methylation of this gene promoter could be a potential target for modifying IL-7-mediated T cell development and survival.     

A new subset of human naive CD8+ T cells defined by low expression of IL-7R alpha

Concomitant with an increased number of memory-type cells, the amount of naive T cells steadily declines with age. Although the regulatory mechanisms behind this conversion are not fully understood, the suggestion is that both alterations in thymic output and homeostatic signals mold the naive T cell pool. In this study, we identify a new subset of circulating CD27(high)CD45RA(high) CD8+ T cells characterized by low IL-7Ralpha message and protein expression. Analysis of TCR repertoire and TCR excision circle content together with ex vivo recovery of IL-7Ralpha expression indicated that these cells should be placed into the naive T cell pool. Compared with conventional IL-7Ralpha(high) naive T cells, this subset displayed significantly lower levels of CD28 and higher levels of HLA-DR. Proliferative responses to anti-CD3/CD28 mAbs were indistinguishable from conventional naive T cells, but the responsiveness to IL-7 was limited. Strikingly, IL-7Ralpha(low) naive T cells were particularly increased in circumstances of naive CD8+ T cells shortage, as in the elderly, in patients early after hemopoietic stem cell transplantation, and in HIV-infected individuals. As common gamma chain cytokines induce rapid down-regulation of IL-7Ralpha, we propose that this new subset of naive T cells may encompass cells that have recently received homeostatic signals.     

Cutting edge: IL-7-independent regulation of IL-7 receptor alpha expression and memory CD8 T cell development

Expression of IL-7Ralpha on a subset of Ag-specific effector CD8 T cells is believed to identify memory cell precursors. However, whether IL-7 regulates IL-7Ralpha expression in vivo and is responsible for selective survival of IL-7Ralpha(+) effector cells is unknown. Our results show that in the absence of IL-7, IL-7Ralpha expression was extinguished on the majority of CD8 T cells responding to virus infection, sustained on a subset of effector cells transitioning to memory, and expressed at high levels by memory cells. Additionally, an IL-7-deficient environment was capable of supporting bcl-2 up-regulation and memory cell development in response to virus infection. Thus, IL-7Ralpha regulation occurs independently of IL-7 in responding CD8 T cells, indicating that CD8 memory T cell precursors are not selected by IL-7/IL-7Ralpha interactions.     

IL-7/anti-IL-7 mAb complexes restore T cell development and induce homeostatic T Cell expansion without lymphopenia

IL-7, a member of the common gamma-chain family of cytokines, is essential for B and T lymphocyte development and homeostasis of mature T cell subsets. Thus, naive and memory T cells are both dependent on IL-7 for survival and homeostatic proliferation under lymphopenic conditions. In line with prior findings with IL-2, we show in this study that the biological activity of IL-7 in vivo is greatly increased by association with anti-IL-7 mAb. Under in vivo conditions, IL-7/mAb complexes displayed 50- to 100-fold higher activity than free IL-7 and induced massive expansion of pre-B cells. IL-7/mAb complexes also increased thymopoiesis in normal mice and restored thymopoeisis in IL-7-deficient mice. For mature T cells, IL-7/mAb complexes induced marked homeostatic proliferation of both naive and memory CD4(+) and CD8(+) cell subsets even under normal T cell-replete conditions. Finally, IL-7/mAb complexes were able to enhance the magnitude of the primary response of Ag-specific naive CD8(+) cells. The strong stimulatory activity of IL-7/mAb complexes could be useful for treatment of immunodeficiency and cancer.     

Homeostasis of naive and memory CD4+ T cells: IL-2 and IL-7 differentially regulate the balance between proliferation and Fas-mediated apoptosis

Cytokines play a crucial role in the maintenance of polyclonal naive and memory T cell populations. It has previously been shown that ex vivo, the IL-7 cytokine induces the proliferation of naive recent thymic emigrants (RTE) isolated from umbilical cord blood but not mature adult-derived naive and memory human CD4(+) T cells. We find that the combination of IL-2 and IL-7 strongly promotes the proliferation of RTE, whereas adult CD4(+) T cells remain relatively unresponsive. Immunological activity is controlled by a balance between proliferation and apoptotic cell death. However, the relative contributions of IL-2 and IL-7 in regulating these processes in the absence of MHC/peptide signals are not known. Following exposure to either IL-2 or IL-7 alone, RTE, as well as mature naive and memory CD4(+) T cells, are rendered only minimally sensitive to Fas-mediated cell death. However, in the presence of the two cytokines, Fas engagement results in a high level of caspase-dependent apoptosis in both RTE as well as naive adult CD4(+) T cells. In contrast, equivalently treated memory CD4(+) T cells are significantly less sensitive to Fas-induced cell death. The increased susceptibility of RTE and naive CD4(+) T cells to Fas-induced apoptosis correlates with a significantly higher IL-2/IL-7-induced Fas expression on these T cell subsets than on memory CD4(+) T cells. Thus, IL-2 and IL-7 regulate homeostasis by modulating the equilibrium between proliferation and apoptotic cell death in RTE and mature naive and memory T cell subsets.     

Chemokine-guided CD4+ T cell help enhances generation of IL-6RalphahighIL-7Ralpha high prememory CD8+ T cells

CD4(+) T cells promote effective CD8(+) T cell-mediated immunity, but the timing and mechanistic details of such help remain controversial. Furthermore, the extent to which innate stimuli act independently of help in enhancing CD8(+) T cell responses is also unresolved. Using a noninfectious vaccine model in immunocompetent mice, we show that even in the presence of innate stimuli, CD4(+) T cell help early after priming is required for generating an optimal pool of functional memory CD8(+) T cells. CD4(+) T cell help increased the size of a previously unreported population of IL-6Ralpha(high)IL-7Ralpha(high) prememory CD8(+) T cells shortly after priming that showed a survival advantage in vivo and contributed to the majority of functional memory CD8(+) T cells after the contraction phase. In accord with our recent demonstration of chemokine-guided recruitment of naive CD8(+) T cells to sites of CD4(+) T cell-dendritic cell interactions, the generation of IL-6Ralpha(high)IL-7Ralpha(high) prememory as well as functional memory CD8(+) T cells depended on the early postvaccination action of the inflammatory chemokines CCL3 and CCL4. Together, these findings support a model of CD8(+) T cell memory cell differentiation involving the delivery of key signals early in the priming process based on chemokine-guided attraction of naive CD8(+) T cells to sites of Ag-driven interactions between TLR-activated dendritic cells and CD4(+) T cells. They also reveal that elevated IL-6Ralpha expression by a subset of CD8(+) T cells represents an early imprint of CD4(+) T cell helper function that actively contributes to the survival of activated CD8(+) T cells.     

CD11chigh dendritic cell ablation impairs lymphopenia-driven proliferation of naive and memory CD8+ T cells

The peripheral lymphocyte pool size is governed by homeostatic mechanisms. Thus, grafted T cells expand and replenish T cell compartments in lymphopenic hosts. Lymphopenia-driven proliferation of naive CD8+ T cells depends on self-peptide/MHC class I complexes and the cytokine IL-7. Lymphopenia-driven proliferation and maintenance of memory CD8+ T cells are MHC independent, but are believed to require IL-7 and contact with a bone marrow-derived cell that presents the cytokine IL-15 by virtue of its high affinity receptor (IL-15Ralpha). In this study we show that optimal spontaneous proliferation of grafted naive and memory CD8+ T cells in mice rendered lymphopenic through gene ablation or irradiation requires the presence of CD11chigh dendritic cells. Our results suggest a dual role of CD11chigh dendritic cells as unique APC and cytokine-presenting cells.     

The human B cell response to IL-13 is dependent on cellular phenotype as well as mode of activation.

Normal mature quiescent human B lymphocytes, isolated as a function of buoyant density, require activation for up-regulation of IL-13R constituents. Cell activation through a combination of surface Ig and CD40 receptor ligation leads to the most substantial message production for IL-13Ralpha1. Functional consequences of this receptor variation, in initially quiescent cells, includes demonstrable effects on cellular proliferation in response to ligand exposure. Variations in the method of surface activation, with particular emphasis on the CD40 receptor, reveals that immobilized CD40 ligand may be sufficient, in and of itself, to up-regulate IL-13Ralpha1, which may bear significance for B-lymphocyte bystander proliferation. Regulation of the IL-13Ralpha1 protein and message also differs as a function of cellular phenotype. Although values are greater in memory than naive B cells, as they are initially isolated from extirpated tonsils, variations in the magnitude of message and protein, as a function of surface stimulation, are more substantial in the naive subset. The magnitude of variation in message production in naive cells is associated with a more vigorous proliferative response to IL-13 than seen in memory lymphocytes. The cellular response to IL-13, as a function of activation and phenotype, is the converse of that demonstrated for IL-2. Evaluation of proliferation, receptor message, ligand binding protein production, and the response to putatively synergistic cytokines reveals that IL-2 is the predominant lymphokine utilized by memory cells. This is in contradistinction to IL-13, which along with IL-4, are the predominant moieties for naive lymphocytes.     

Classical ataxia telangiectasia patients have a congenitally aged immune system with high expression of CD95

Ataxia-telangiectasia (A-T) is a rare neurodegenerative immunodeficiency disorder caused by mutations in the ataxia telangiectasia mutated gene. Patients commonly have lymphopenia and Ig-production abnormalities. We used multicolor flow cytometry and IL-7 ELISA to investigate the effect of A-T and age on the proportions of major lymphocyte subsets and their pattern of CD95 expression in relation to IL-7 levels in 15 classical A-T patients. We also analyzed the sensitivity of T cells from four classical A-T patients to CD95-mediated apoptosis using TUNEL and caspase-activation assays. Our results confirmed lymphopenia and a deficiency in naive T and B cells in A-T patients. In contrast to controls, the proportions of naive and memory T and B cell subsets in A-T patients did not vary in relation to age. There was no evidence of a deficiency in plasma IL-7 or IL-7R expression, and IL-7 concentration correlated positively with CD95 expression on CD4(+) T cells. CD95 expression on unstimulated A-T lymphocytes was high, and the apoptotic sensitivity of activated naive and central memory T cells was increased. These findings show that the immunodeficiency in A-T patients may be described as congenitally aged and is not progressive. The naive cell deficiency is not related to a deficiency in IL-7 or its receptor. However, IL-7 may upregulate CD95 on A-T lymphocytes. High CD95 expression and increased apoptotic sensitivity of activated naive and central memory T cells may result in an increased level of CD95-mediated apoptosis, which could contribute to the congenital lymphopenia in A-T.     

Antigen controls IL-7R alpha expression levels on CD8 T cells during full activation or tolerance induction

The high-affinity chain of the IL-7 receptor, IL-7Ralpha (CD127), is expressed by effector CD8 T cells that have the capacity to become memory cells. IL-7Ralpha expression is uniformly high on naive CD8 T cells, and the majority of these cells down-regulate expression upon antigenic challenge. At the peak of expansion, the fraction of effectors expressing high IL-7Ralpha varies depending on the response examined. The signals that a CD8 T cell receives during a response to Ag that lead to altered expression of IL-7Ralpha have not been fully defined. In vitro experiments demonstrated that Ag alone is sufficient to down-regulate IL-7Ralpha on all cells and most of the cells rapidly re-express the receptor upon removal from Ag. Expression was not altered by the B7.1 costimulatory ligand or when IL-12 was present to provide the signal needed for development of effector functions, indicating that TCR engagement is sufficient to regulate IL-7Ralpha expression. Consistent with this, in vivo priming with peptide Ag resulted in IL-7Ralpha expression that inversely correlated with Ag levels, and expression levels were not changed when IL-12 or adjuvant were administered with Ag. A large fraction of the cells present at the peak of expansion had re-expressed IL-7Ralpha, but most of these cells failed to survive; those that did survive expressed high IL-7Ralpha levels. Thus, Ag-dependent signals regulate IL-7Ralpha levels on responding CD8 T cells, and this occurs whether the responding cells become fully activated or are rendered tolerant by administration of peptide Ag alone.     

Constitutive expression of IL-7 receptor alpha does not support increased expansion or prevent contraction of antigen-specific CD4 or CD8 T cells following Listeria monocytogenes infection

Expression of IL-7Ralpha (CD127) has been suggested as a major determinant in the survival of memory T cell precursors. We investigated whether constitutive expression of IL-7Ralpha on T cells increased expansion and/or decreased contraction of endogenous Ag-specific CD4 and CD8 T cells following infection with Listeria monocytogenes. The results indicate that constitutive expression of IL-7Ralpha alone was not enough to impart an expansion or survival advantage to CD8 T cells responding to infection, and did not increase memory CD8 T cell numbers over those observed in wild-type controls. Constitutive expression of IL-7Ralpha did allow for slightly prolonged expansion of Ag-specific CD4 T cells; however, it did not alter the contraction phase or protect against the waning of memory T cell numbers at later times after infection. Memory CD4 and CD8 T cells generated in IL-7Ralpha transgenic mice expanded similarly to wild-type T cells after secondary infection, and immunized IL-7Ralpha transgenic mice were fully protected against lethal bacterial challenge demonstrating that constitutive expression of IL-7Ralpha does not impair, or markedly improve memory/secondary effector T cell function. These results indicate that expression of IL-7Ralpha alone does not support increased survival of effector Ag-specific CD4 or CD8 T cells into the memory phase following bacterial infection.     

In vivo differentiated cytokine-producing CD4(+) T cells express functional CCR7

Chemokines and their receptors fulfill specialized roles in inflammation and under homeostatic conditions. CCR7 and its ligands, CCL19 and CCL21, are involved in lymphocyte recirculation through secondary lymphoid organs and additionally navigate lymphocytes into distinct tissue compartments. The role of CCR7 in the migration of polarized T effector/memory cell subsets in vivo is still poorly understood. We therefore analyzed murine and human CD4(+) cytokine-producing cells developed in vivo for their chemotactic reactivity to CCR7 ligands. The responses of cells producing cytokines, such as IFN-gamma, IL-4, and IL-10, as well as of subsets defined by memory or activation markers were comparable to that of naive CD4(+) cells, with slightly lower reactivity in cells expressing IL-10 or CD69. This indicates that CCR7 ligands are able to attract naive as well as the vast majority of activated and effector/memory T cell stages. Chemotactic reactivity of these cells toward CCL21 was absent in CCR7-deficient cells, proving that effector cells do not use alternative receptors for this chemokine. Th1 cells generated from CCR7(-/-) mice failed to enter lymph nodes and Peyer's patches, but did enter a site of inflammation. These findings indicate that CD4(+) cells producing effector cytokines upon stimulation retain the capacity to recirculate through lymphoid tissues via CCR7.     

Combined IL-15/IL-15Ralpha immunotherapy maximizes IL-15 activity in vivo

IL-15 has substantial potential as an immunotherapeutic agent for augmenting immune responses. However, the activity of IL-15 is mediated by a unique mechanism in which the cytokine is transpresented by cell-bound high-affinity IL-15Ralpha to target cells expressing the IL-15Rbeta and the common gamma-chain. Thus, the efficacy of administered IL-15 alone may be limited by the availability of free IL-15Ralpha. We now show that administration of soluble IL-15/IL-15Ralpha complexes greatly enhanced IL-15 half-life and bioavailability in vivo. Treatment of mice with this complex, but not with IL-15 alone, resulted in robust proliferation of memory CD8 T cells, NK cells, and NK T cells. The activity of the complex required IL-15Rbeta, but not IL-15Ralpha, expression by the responding cells and was IL-7-independent. Interestingly, IL-15/IL-15Ralpha immunotherapy also caused naive CD8 T cell activation and development into effector cells and long-term memory T cells. Lastly, complexed IL-15, as compared with IL-15 alone, dramatically reduced tumor burden in a model of B16 melanoma. These findings hold significant importance for the use of IL-15 as a potential adjuvant/therapeutic and inducer of homeostatic proliferation, without the necessity for prior immunodepletion.     

Cutting edge: requirement for IL-15 in the generation of primary and memory antigen-specific CD8 T cells

IL-15 and IL-15Ralpha are required for generation of memory-phenotype CD8 T cells in unimmunized mice. However, the role of IL-15 in primary expansion and generation of Ag-specific memory CD8 T cells in vivo has not been investigated. We characterized the CD8 T cell response against vesicular stomatitis virus (VSV) in IL-15(-/-) and IL-15Ralpha(-/-) mice. Surprisingly, IL-15 was required for primary expansion of VSV-specific CD8 T cells. The generation of VSV-specific memory CD8 T cells was also impaired without IL-15 signaling, and this defect correlated with a decrease in memory CD8 T cell turnover. Despite minimal proliferation without IL-15, a subset of memory cells survived long-term. IL-15Ralpha expression was low on naive CD8 T cells, up-regulated on Ag-specific effector cells, and sustained on memory cells. Thus, IL-15 was important for the generation and the subsequent maintenance of antiviral memory CD8 T cells.     

Loss of IL-7 receptor alpha on CD4+ T cells defines terminally differentiated B cell-helping effector T cells in a B cell-rich lymphoid tissue

IL-7 plays important roles in development and homeostatic proliferation of lymphocytes. IL-7 uses a receptor composed of IL-7Ralpha (CD127) and the common gamma-chain (CD132) to transmit its signal. It has been unknown how CD127 is regulated during Th cell differentiation to the B cell-helping T cell lineage. In this study, we report that loss of CD127 defines terminally differentiated B cell-helping effector T cells in human tonsils. Although naive CD4(+) T cells uniformly express CD127, the memory/effector (non-FOXP3(+)) CD4(+) T cells are divided into CD127(+) and CD127(-) cells. The CD127(-) T cells are exclusively localized within the germinal centers where B cells become plasma and memory B cells, whereas CD127(+) T cells are found in T cell areas and the area surrounding B cell follicles. Consistently, the CD127(-) T cells highly express the B cell zone homing receptor CXCR5 with concomitant loss of CCR7. Compared with CD127(+) memory T cells, CD127(-) T cells have considerably shorter telomeres, do not proliferate in response to IL-7, and are prone to cell death. The CD127(-) T cells produce a large amount of the B cell follicle-forming chemokine CXCL13 upon stimulation with B cells and Ags. Most importantly, they are highly efficient in helping B cells produce Igs of all isotypes in a manner dependent on CD40L and ICOS and inducing activation-induced cytidine deaminase and Ig class switch recombination. The selective loss of CD127 on the B cell-helping effector T cells would have implications in regulation and termination of Ig responses.     

Cutaneous lymphocyte antigen expression on activated lymphocytes and its association with IL-12R (beta1 and beta2), IL-2Ralpha, and CXCR3

The majority of T lymphocytes that infiltrate psoriatic lesions express cutaneous lymphocyte antigen (CLA), a skin homing receptor involved in the influx of memory T cells to cutaneous sites. We investigated CLA expression on normal human peripheral blood mononuclear cells (PBMCs) and evaluated its association with IL-12 receptors, chemokine receptor, CXCR3, and IL-2Ralpha. PBMCs were stimulated in vitro with or without polyclonal activators (mitogen, or superantigens, or anti-CD3+anti-CD28) in the presence or absence of exogenous rhIL-12. The percentage of CLA+ T lymphocytes increased significantly after superantigen stimulation compared to anti-CD3+anti-CD28 or mitogen activation. The majority of activation induced CLA+ T lymphocytes co-expressed IL-12Rbeta1, IL-12Rbeta2, CXCR3, and CD25 in the presence of rhIL-12. Our results indicate that CLA expression on activated T lymphocytes is IL-12 and activation dependent and correlates with the expression of IL-12 receptors, IL-2Ralpha, and CXCR3. Monitoring the levels of Th1 differentiation markers such as CXCR3 and IL-12Rbeta2 along with activation marker, CD25 on skin homing CLA+ T lymphocytes may provide insight into the mechanism of action of immunotherapies directed against Th1 type skin inflammatory diseases.