IL-7Rα Chain Research Articles

The Role of Thymic Stromal Lymphopoietin in CD8+ T Cell Homeostasis

Thymic stromal lymphopoietin (TSLP) is a cytokine produced by stromal cells, epithelial cells, and basophils that acts on dendritic cells, mast cells, and CD4(+) T cells. The receptor for TSLP contains a TSLP-specific receptor chain (TSLPR) and the IL-7R alpha-chain. Although IL-7 critically controls the expansion and survival of naive and memory CD8(+) T cells, an action for TSLP on CD8(+) T cells has not been reported. We now demonstrate that CD8(+) T cells express TSLPR and that TSLP activates both STAT5 and Akt and induces Bcl-2 in these cells. Correspondingly, TSLP increases CD8(+) T cell survival in vitro as well as in wild-type and T-depleted mice in vivo, without altering the homeostatic proliferation of these cells. Moreover, TSLP can maintain CD8(+) T cells even in the absence of IL-7. Thus, our data reveal that TSLP contributes to CD8(+) T cell homeostasis in both normal and lymphopenic conditions.

Early Human B-Lineage Cells Can Make Functional Igκ Gene Rearrangements and Express Igκ Protein in the Absence of Functional VDJH Rearrangements.

One of the tools available to study human B cell development are xenogeneic murine stromal cells that serve as a microenvironment to support development of B-lineage cells from human hematopoietic stem cells. Efforts to clarify the role of IL-7 signaling in human B cell development have been rejuvenated by the observation that cord blood CD34+ stem cells cultured on murine MS-5 stromal cells develop into CD19+ B-lineage cells expressing the IL-7R (i.e., IL-7Rα chain, CD127 and gamma common chain, CD132), and that signaling through IL-7R is essential for development of CD19+ B-lineage cells in this model. CD19+/CD127+ cells have survival attributes and patterns of Bcl-2 and p27KIP1 expression that are distinct from CD19+/CD127− cells. CD127+ cells are Bcl-2hi, p27KIP1lo and CD127− cells are Bcl-2lo, p27KIP1hi. However, the relationship between IL-7R expression/signaling and the onset/progression of IgH and IgL rearrangement patterns in early human B-lineage cells is unknown. We hypothesized that IL-7R expression would signify the potential for IgH gene rearrangement and eventually IgL gene rearrangement, whereas absence of expression would signify failure to make in-frame rearrangements. To test this hypothesis, CD19+ cells from three independent 4-wk cord blood/MS-5 xenogeneic cultures were FACS-purified into CD19+/CD127+ and CD19+/CD127− fractions, and a quantitative real time PCR assay was employed to characterize IgH and IgL rearrangements at the level of the sorted populations. The majority of cells in both fractions had incomplete DJH rearrangements. However, < 10% of CD127+ and CD127− cells had complete VDJH rearrangements and those that did primarily used VH1, 2 and 3 families. This data was in agreement with immunofluorescent staining which showed that ~ 5% of CD127+ and CD127− cells expressed cytoplasmic Igμ heavy chains. Neither elimination of IL-7 signaling nor addition of exogenous IGF-1 promoted the appearance of cells expressing Igμ heavy chains. Thus, many of the early B-lineage cells that developed in this model had an IgH rearrangement profile that resembled pre-B-I cells isolated from pediatric marrow. Unexpectedly, analysis of IgL gene rearrangements in the CD19+/CD127+ and CD19+/CD127− fractions showed profound differences. CD127+ cells harbored very few rearrangements at the Igκ and Igλ light chain loci (estimated to be < 5% of cells). However, CD127− cells showed extensive VJκ and VJλ rearrangements, as well as evidence that the kappa deleting element had undergone rearrangement with Vκ genes and the intron recombination signal sequence. Thus, despite having only a small percentage of cells with complete VDJH rearrangements, the CD127− fraction showed a high level of recombinatorial activity in the light chain loci. Many of the VJκ rearrangements were in-frame, since Igκ protein was detected in the majority of CD127− cells by flow cytometry, immunohistochemistry, and Western blotting. These results show that in-frame rearrangement at the IgH locus is not a requirement for developing human B-lineage cells to initiate rearrangement at the IgL loci. They further suggest that the low frequency of cells with complete VDJH rearrangements reflects epigenetic silencing of VH gene clusters, or the absence of a stimulus in the xenogeneic culture that induces VDJH rearrangements. The existence of cells expressing Igκ light chain protein in the absence of Igμ heavy chain protein may represent a heretofore unrecognized population of human B-lineage cells with heightened recombinatorial flexibility and/or a population destined to die in the marrow.

Peripheral Alloantigen Drives Early Dysfunction and Eventual Exhaustion of CTL Following Delayed Donor Leukocyte Infusions.

T cell immunotherapies for cancer should ideally generate high levels of anti-tumor activity, with minimal host injury and permit the prolonged survival of functional memory/effector cells to prevent tumor recurrence. Following allogeneic stem cell transplantation, delayed donor leukocyte infusion (DLI) is one strategy employed to induce graft-versus-leukemia (GVL) responses while limiting the risk of host injury in terms of graft-versus-host disease. However, patients remain at significant risk of relapse following DLI and murine models of delayed DLI indicate that this results from the eventual loss of functional, alloreactive cytotoxic T lymphocytes (CTL) [Mapara et al. Transplantation 2003]. We hypothesised that the loss of functional CTL is driven by persistent stimulation of donor CD8 cells by alloantigen expressed by peripheral tissues. In order to follow and characterise an alloreactive CD8 response under conditions in which alloantigen was present or absent in peripheral tissues, we employed a model in which either parental B6 (H2b) or B6 x DBA-2 F1 (BDF1, H2dxb) mice were lethally irradiated and reconstituted with a mixture of B6 and BDF1 T cell depleted bone marrow. 8-10 weeks later congenic CD45.1 B6 splenocytes were transferred into the established mixed chimeras. This allowed us to test the importance of peripheral antigen in the loss of alloreactive CTL responses, since alloantigen was either restricted to the hematopoietic system (B6 +BDF1 → B6) or was ubiquitously expressed (B6 +BDF1 → BDF1). Following transfer of CD45.1 B6 splenocytes, the ensuing alloantigenspecific T cell response in both groups led to the elimination of alloantigen-positive (BDF1-derived) hematopoietic elements. Thereafter, alloreactive CD8 cells resided in an environment in which peripheral alloantigen was present (PA+) or absent (PA-). We observed similar kinetics of initial CD45.1+ CD8 cell proliferation and expansion and similar acquisition of a CD44highCD62Llow phenotype. However, by day 60, there were striking differences in the phenotype and function of transferred CD8 cells. In PA- hosts, CD45.1+ CD8 cells killed allogeneic target cells effectively both in vitro and in vivo, underwent rapid proliferation in a mixed leukocyte reaction and produced the effector cytokine, IFN-γ. In contrast CD45.1+ CD8 cells from PA+ hosts had little or no cytotoxic activity, did not proliferate to alloantigen and were IFN-γlow. Moreover, CD45.1+ CD8 cells from PA+ hosts displayed high levels of the co-inhibitory receptor PD-1, low levels of the IL-7Rα chain and responded poorly to IL-7 and IL-15 in vitro, a phenotype typical of the ‘exhaustion’ signature observed in CTL following chronic antigen exposure. In comparison, CD45.1+ CD8 cells from PA- hosts expressed significantly lower levels of PD-1, higher levels of IL-7Rα and demonstrated better responsiveness to IL-7 and IL-15 in vitro. In vitro PD-1 or PD-L1 blockade restored IFN-γ generation to CD45.1+ CD8 cells from PA+ hosts, suggesting that the PD-1 pathway may play a functional role in driving exhaustion of these cells. Importantly we observed no loss of long-term alloreactive CD4 responses in either PA+ or PA- hosts. This finding is consistent with a model in which peripheral alloantigen drives exhaustion since the majority of cells expressing Class II alloantigens in PA+ and PA- hosts would be restricted to the hematopoietic system and thus, would have been cleared in the initial alloresponse. The full exhausted phenotype of alloreactive CD8 cells described above was not seen until at least 30 days after transfer to PA+ hosts. However, as early as day 14, CTL primed in PA+ hosts produced less IFN-γ in comparison to those primed in PA-hosts, even though they were still equivalent in terms of their cytotoxicity. Furthermore, when CD8 cells primed in PA+ hosts were transferred to secondary antigen-free hosts, they still displayed reduced ‘fitness’ compared to CTL originally primed in PA- hosts. These data show that peripheral alloantigen qualitatively affects donor CTL function during priming and drives their eventual exhaustion. Additionally they suggest that blockade of co-inhibitory signals may have potential in restoring function to such cells as has been demonstrated in models of chronic infection.

Tardy DCs to the (Th1) rescue

In newborns, the sluggish appearance of one cell population means death to another, according to Lee et al. on page 2269. These results may help explain why newborns are highly susceptible to certain infections. Figure 1 Delayed development of interferon-γ–producing Th1 cells in newborns. Newborn mice exposed to antigen respond by activating both T helper (Th)-1 and Th2 cells. Yet a second exposure causes allergy-promoting Th2 cells to thrive but microbe-fighting Th1 cells to die. Previous work by this group showed that antigen exposure during the first few days of life caused Th1 cells to express the cytokine receptor chain IL-13Rα1, a receptor not commonly found on these cells, which then teamed up with the IL-4Rα chain. The resulting heteroreceptor induced Th1 cell death when triggered by Th2-promoting IL-4 during secondary antigen exposure. At six days of age, the authors now show, Th1 cells had a reversal of fortune. The turning point was marked by the appearance of a subset of antigen-presenting CD8α+ dendritic cells (DCs) that churned out life-saving IL-12. Giving newborns extra IL-12, which is feebly produced before day 6, or providing them with IL-12–producing DCs blunted the expression of IL-13Rα1 and rescued the Th1 cells. What delays the development of this DC population relative to other subsets remains unknown. RB

Glucocorticoids induce an activated, anti-inflammatory monocyte subset in mice that resembles myeloid-derived suppressor cells

Glucocorticoids (GC) are still the most widely used immunosuppressive agents in clinical medicine. Surprisingly, little is known about the mechanisms of GC action on monocytes, although these cells exert pro- and anti-inflammatory effects. We have shown recently that GC induce a specific monocyte phenotype with anti-inflammatory properties in humans. We now investigated whether this also applies for the murine system and how this subset would relate to recently defined murine subtypes. After treatment with dexamethasone for 48 h, monocytes up-regulated scavenger receptor CD163 and Gr-1, down-regulated CX(3)CR1, and shared with human GC-treated monocytes functional features such as low adhesiveness but high migratory capacity. They specifically up-regulated anti-inflammatory IL-10, but not TGF-beta, and in contrast to their human counterparts, they down-regulated IL-6. Although GC-induced monocytes down-regulated CX(3)CR1, a distinctive marker for classical/proinflammatory human and murine monocytes (CX(3)CR1(lo)CCR2(+)Ly6C(hi)), they differed from this physiologically occurring subset, as they remained Ly6C(med) and unactivated (CD62 ligand(++)). In addition to their immunosuppressive effects, they were CD11b(+)Gr-1(+) and expressed the IL-4Ralpha chain (CD124), a recently described, signature molecule of tumor-induced myeloid-derived suppressor cells (MDSC). We therefore generated murine MDSC in B16 melanoma-bearing mice and indeed found parallel up-regulation of CD11b(+)Gr-1(+) and CD124 on GC-induced monocytes and MDSC. These data allow us to speculate that the GC-induced subtype shares with inflammatory monocytes the ability to migrate quickly into inflamed tissue, where they, however, exert anti-inflammatory effects and that similarities between GC-induced monocytes and MDSC may be involved in progression of some tumors observed in patients chronically treated with GC.

MEK1/2 Induces STAT5-Mediated Germline Transcription of the TCRγ Locus in Response to IL-7R Signaling

The IL-7R plays an essential role in gammadelta T cell development by inducing V-J recombination of the TCRgamma locus through STAT5. Although tyrosine residues in the intracellular domain of the mouse IL-7R alpha-chain (IL-7Ralpha) have been implicated in STAT5 activation, it is still unknown whether they are essential for gammadelta T cell development. In this study, we showed that those IL-7Ralpha tyrosine residues are not essential for gammadelta T cell development, because phenylalanine replacement of four intracellular tyrosine residues (IL-7R-FFFF) partially rescued gammadelta T cell development of IL-7Ralpha-/- progenitors. To examine signaling pathways activated by IL-7R-FFFF, we introduced a chimeric receptor consisting of the human IL-4R alpha-chain and mouse IL-7R-FFFF (4R/7R-FFFF) into an IL-7-dependent pre-B cell line and found that 4R/7R-FFFF induced TCRgamma germline transcription and STAT5 activation. Treatment of cells with MEK1/2 inhibitors significantly decreased levels of TCRgamma germline transcription and STAT5 tyrosine phosphorylation mediated by 4R/7R-FFFF, suggesting that MEK1/2 plays an alternative role in STAT5 activation by IL-7R. MEK1/2 associated with STAT5 and induced STAT5 tyrosine phosphorylation and DNA binding activity. Furthermore, MEK1 directly phosphorylated a STAT5 tyrosine residue in vitro. Finally, active MEK1 partially rescued TCRgamma germline transcription by IL-7R in a pre-T cell line. These results demonstrate that MEK1/2 induces TCRgamma germline transcription by phosphorylating STAT5 through IL-7R-FFFF and suggest a potential role for MAPK in IL-7R tyrosine-independent activation of STAT5.

Mutations in CHD7 in patients with CHARGE syndrome cause T–B + natural killer cell + severe combined immune deficiency and may cause Omenn-like syndrome

More than 11 genetic causes of severe combined immunodeficiency (SCID) have been identified, affecting development and/or function of T lymphocytes, and sometimes B lymphocytes and natural killer (NK) cells. Deletion of 22q11.2 is associated with immunodeficiency, although less than 1% of cases are associated with T-B + NK + SCID phenotype. Severe immunodeficiency with CHARGE syndrome has been noted only rarely Omenn syndrome is a rare autosomal recessive form of SCID with erythroderma, hepatosplenomegaly, lymphadenopathy and alopecia. Hypomorphic recombination activating genes 1 and 2 mutations were first described in patients with Omenn syndrome. More recently, defects in Artemis, RMRP, IL7Ralpha and common gamma chain genes have been described. We describe four patients with mutations in CHD7, who had clinical features of CHARGE syndrome and who had T-B + NK + SCID (two patients) or clinical features consistent with Omenn syndrome (two patients). Immunodeficiency in patients with DiGeorge syndrome is well recognized--CHARGE syndrome should now be added to the causes of T-B + NK + SCID, and mutations in the CHD7 gene may be associated with Omenn-like syndrome.

TLR Ligand-Induced Type I IFNs Affect Thymopoiesis

The interactions between TLRs and their ligands have profound immune modulation properties. Attention has focused mostly on the impact of TLR ligands on peripheral innate and adaptive immunity during viral infections, whereas little impact of TLR activation has been shown on thymic development. Here we show that treatment of murine fetal thymic organ cultures (FTOCs) with TLR3 or TLR7 ligands induced rapid expression of IFN-alpha and -beta mRNA, hallmarks of acute and chronic viral infections. This resulted in an early developmental blockade, increased frequencies of apoptotic cells, and decreased proliferation of thymocytes, which led to an immediate decrease in cellularity. FTOCs infected with vesicular stomatitis virus, known to act through TLR7, were similarly affected. Down-regulation of IL-7R alpha-chain expression, together with an increased expression of suppressor of cytokine signaling-1 and a concomitant decreased expression of the transcriptional regulator growth factor independence 1 were observed in TLR ligands or IFN-treated FTOCs. This indicates a role for these pathways in the observed changes in thymocyte development. Taken together, our data demonstrate that TLR activation and ensuing type I IFN production exert a deleterious effect on T cell development. Because TLR ligands are widely used as vaccine adjuvants, their immunomodulatory actions mediated mainly by IFN-alpha suggested by our results should be taken in consideration.

The opposite effects of IL-15 and IL-21 on CLL B cells correlate with differential activation of the JAK/STAT and ERK1/2 pathways

The clonal expansion of chronic lymphocytic leukemia (CLL) cells requires the interaction with the microenvironment and is under the control of several cytokines. Here, we investigated the effect of IL-15 and IL-21, which are closely related to IL-2 and share the usage of the common gamma chain and of its JAK3-associated pathway. We found remarkable differences in the signal transduction pathways activated by these cytokines, which determined different responses in CLL cells. IL-15 caused cell proliferation and prevented apoptosis induced by surface IgM cross-linking. These effects were more evident in cells stimulated via surface CD40, which exhibited increased cell expression of IL-15Ralpha chain and, in some of the cases, also of IL-2Rbeta. IL-21 failed to induce CLL cell proliferation and instead promoted apoptosis. Following cell exposure to IL-15, phosphorylation of STAT5 was predominantly observed, whereas, following stimulation with IL-21, there was predominant STAT1 and STAT3 activation. Moreover, IL-15 but not IL-21 caused an increased phosphorylation of Shc and ERK1/2. Pharmacological inhibition of JAK3 or of MEK, which phosphorylates ERK1/2, efficiently blocked IL-15-induced CLL cell proliferation and the antiapoptotic effect of this cytokine. The knowledge of the signaling pathways regulating CLL cell survival and proliferation may provide new molecular targets for therapeutic intervention.

Interleukin 2: from immunostimulation to immunoregulation and back again

Interleukin 2 (IL-2) was one of the first cytokines to be discovered. However, the complex role of IL-2 and its receptor in the regulation of immune responses is only now emerging. This review explores the various signals triggered by IL-2 and discusses their translation into biological function. A model is outlined that accommodates the seemingly contradictory functions of IL-2, and explains how one cytokine can be an essential T-cell growth and differentiation factor and yet also be indispensable to maintain peripheral tolerance.

Identification of CS1 Peptides for Induction of Antigen-Specific CTLs in Multiple Myeloma.

Despite progress in the treatment of multiple myeloma (MM), the disease remains incurable. Therefore novel therapeutic approaches are required to achieve better outcome in patients. Development of peptide-based immunotherapy against tumor specific or associated antigens offers an attractive approach that may lead to tumor-targeted cellular therapy in MM patients. Recently, CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family, was found to be highly expressed by the tumor cells of the majority of MM patients but not other normal tissues. Importantly, a novel anti-CS1 HuLuc63 mAb induced significant anti-myeloma killing in vitro and in vivo (Abstract #950059). In addition, CS1 may play a role in MM cell survival (Abstract #953659). In this study, we asked whether CS1 is a suitable myeloma-associated antigen for cellular therapy in MM. We wanted to identify potential immunogenic peptides derived from CS1 and tested whether these peptides evoke MM-specific cytotoxic T lymphocytes (CTLs). First, we predicted the potential peptide sequences of CS1 that could bind to HLA-A*0201 molecule using three databases (RANKPEP, BIMAS and NetMHC). A total of four peptides were selected and synthesized according to the high binding scores among all the databases. Next, peptide-T2 cells binding assay confirmed these peptides with high binding affinity to HLA-A*0201 molecule (Fluorescence index: 2.19, 3.28, 3.44 and 3.49). To generate the peptide-specific CTLs, HLA-A*0201-positive normal human CD8+ T lymphocytes were stimulated with autologuous dendritic cells (DCs) or T2 cells pulsed with candidate CS1-peptides. The CTL cell lines were established after several rounds restimulation. We have evaluated the immunogenicity of the expanded CTLs by the stimulation with peptide-pulsed or unpulsed T2 cells. Our data demonstrated that one peptide-induced CTLs (CS1-CTLs) possessed high antigen-specific immune responses with the HLA-A*0201-restriction relative to a certain level of immune responses displayed by another three peptide-induced CTLs. We further evaluated the proliferative activity of CS1-CTLs by 3H-TdR incorporation assay. Upon stimulation by peptide-pulsed T2 cells, the cell proliferation of CS1-specific CTLs increased approximately 55-fold, compared with unstimulated cells (pulsed vs. unpulsed: 22894 vs. 423 cpm). The activation of CS1-CTLs was monitored by the expression of CD25 (IL-2Rα chain) on peptide-stimulated CS1-CTLs (%CD25+ cells: pulsed vs. unpulsed: 98% vs. 13%). CS1-CTLs significantly secreted 14-fold higher levels of IFN- γ in response to peptide-pulsed T2 cells (pulsed vs. unpulsed: 615 vs. 46 pg/ml). Importantly, the expanded CS1-CTLs generated high cytotoxicity (93%) in response to peptide pulsed T2 cells. Furthermore, more than 30% of killing activity by CS1-CTLs against HLA-A*0201+CS1+ MCCAR cells was observed at the effector: target ratio of 20:1. In contrast, these CS1-CLTs did not kill HLA-A*0201+/CS1− U266 and HLA-A*0201−/CS1+ MM1S target cells. These results support CS1 as an antigenic target for the induction of peptide-specific CTLs against MM cells. Further functional studies are underway to confirm potential CS1-specific cellular therapy to treat MM.

CD127 Expression Confers Enhanced Survival on Human B-Lineage Cells; Implications for the Role of IL-7 Signaling in Human B Lymphopoiesis.

The IL-7Rα chain (CD127) is expressed on some, but not all, CD19+/surface μ-B-lineage cells isolated from fetal and adult marrow, and umbilical cord blood, but the functional consequences of CD127 expression in early human B cell development during all stages of human life are unresolved. We have described a xenogeneic culture model for generating CD19+ B-lineage cells from CD34+ cord blood stem cells following a 4 wk co-culture on murine MS-5 stromal cells (Johnson SE et al., 2005 J Immunol.). The majority of these cells express a pro-B cell phenotype i.e., CD19+/CD10+/CD20lo/CD21−/CD22+/CD24+/pre-BCR-. Moreover, 1–4% express surface μ heavy chains (HC), and some express κ or λ light chains. We have also reported that 20–40% of CD19+ cells emerging in the xenogeneic culture express CD127, CD127+ cells are more blastic than CD127- cells, and development of CD19+ cells is substantially blocked in xenogeneic cultures treated with anti-murine IL-7 (Johnson SE, ibid). The goal of the present study was to further characterize the developmental potential and patterns of gene expression in CD19+/CD127+ and CD19+/CD127− cells that emerge in xenogeneic cultures. We hypothesized that CD127 expression defines a subset of CD19+ B cell precursors with heightened potential to become mature B-lymphocytes. CD19+/CD127+ and CD19+CD127− populations that emerged in 4 wk xenogeneic cultures were sorted to > 95% purity by FACS. Immunofluorescent staining showed that surface and intracellular μ HC positive cells were present in both sorted populations at frequencies of 3–4%. Using an RT-PCR-based sequencing approach, we analyzed approximately 100 IgM V region sequences from both CD19+/CD127+ and CD19+/CD127− FACS-purified populations. The results indicated that the sequences from both populations were:1.non-mutated and used VH4, D and J gene segments that were similar to what would be expected in a cord blood B cell repertoire, and2.exhibited no statistically significant difference in N segment length, CDR3 length, or CDR3 charges.These results suggest that the amplified sequences were likely derived from the 3–4% cytoplasmic μ HC+ pre-B cells present in both sorted populations. The immunofluorescent staining and VDJH rearrangement sequence results suggest that large CD19+/CD127+/cytoplasmic μ HC+ pre-B cells may differentiate into small CD19+/CD127−/cytoplasmic μ HC+ cells in the xenogeneic culture. This was further supported by showing that intra-tibial injection of FACS-purified CD19+/CD127+ and CD19+/CD127− cells into sub-lethally irradiated NOD-SCID mice led to the appearance of surface μ HC+ cells from both sorted populations. Western blotting of sorted CD19+/CD127+ and CD19+/CD127− cells showed the presence of Bcl-2, p-GSK3β (specifically p-Ser9, the residue phosphorylated by AKT) and pERK1/2 in CD19+/CD127+ cells, while these proteins/phosphoproteins were undetectable in CD19+/CD127− cells. By contrast, expression of the cell cycle inhibitor p27KIP1 was elevated in CD19+/CD127− cells compared to CD19+/CD127+ cells. Furthermore, FACS-purified CD19+/CD127+ cells survived longer than CD19+/CD127− cells when cultured in the absence of MS-5, and the survival of the former was enhanced by stimulation with IL-7. The collective results indicate that CD127 expression identifies a population of normal human CD19+ B cell precursors with a pattern of gene expression and survival/proliferation attributes consistent with a crucial role in the development of the B-lymphocyte arm of the human immune system.

Dual Roles of IL-15 in Maintaining IL-7RαlowCCR7− Memory CD8+ T Cells in Humans via Recovering the Phosphatidylinositol 3-Kinase/AKT Pathway

Recently, we identified two subsets of CCR7(-) memory CD8(+) T cells expressing high and low levels of the IL-7R alpha-chain (IL-7Ralpha) that is essential for memory T cell survival in human peripheral blood. IL-7Ralpha(low)CCR7(-) memory CD8(+) T cells that produce effector cytokines and perforin have impaired proliferation and survival in response to TCR triggering and IL-7, respectively. These findings raise a question of how such cells are sustained at significant numbers, >20% of peripheral CD8(+) T cells, despite impaired IL-7- and TCR-mediated cell maintenance. In this study, we demonstrate that IL-7Ralpha(low)CCR7(-) memory CD8(+) T cells have increased expression of IL-2/15R beta-chain (IL-2/15Rbeta), which is critical for IL-15 signaling, with enhanced gene expression of T box expressed in T cells (T-bet) and eomesodermin (eomes), transcriptional factors involved in IL-2/15Rbeta expression compared with IL-7Ralpha(high)CCR7(-) memory CD8(+) T cells. Such a cytokine chain is functional as IL-7Ralpha(low)CCR7(-) memory CD8(+) T cells proliferate considerably in response to IL-15. Furthermore, adding IL-15 to TCR triggering recovers impaired TCR-mediated proliferation of IL-7Ralpha(low) memory CD8(+) T cells via restoring the activation of the PI3K/AKT pathway. These findings indicate that IL-15 has dual roles in maintaining IL-7Ralpha(low)CCR7(-) memory CD8(+) T cells via TCR-dependent and -independent mechanisms. Moreover, IL-15 can be useful in reviving impaired proliferative function of such memory CD8(+) T cells with effector functions against infections and tumors via rescuing the PI3K/AKT pathway.

Thymic Stromal-Derived Lymphopoietin Induces Proliferation of Pre-B Leukemia and Antagonizes mTOR Inhibitors, Suggesting a Role for Interleukin-7Rα Signaling

Understanding the pathogenesis of leukemia in the context of lymphopoiesis may reveal novel therapeutic targets. Previously, we have shown that mTOR inhibitors (MTI) show activity in vitro and in preclinical models of both human and murine precursor B acute lymphoblastic leukemia (pre-B ALL), inhibiting cell proliferation and inducing apoptosis. These MTI-mediated effects can be reversed by interleukin-7 (IL-7), an important regulator of early B-cell development. This observation led us to examine the contribution of signaling via the IL-7Ralpha chain, which is shared by the receptor complexes of IL-7 and thymic stromal-derived lymphopoietin (TSLP). TSLP is closely related to IL-7 and active in lymphopoiesis, but an effect of TSLP on leukemia cells has not been described. We examined the effect of TSLP on pre-B ALL cells and their response to MTIs. Here, we show that TSLP stimulates proliferation of pre-B ALL cell lines. TSLP also partially reverses the effects of MTI on proliferation, apoptosis, and ribosomal protein S6 and 4E-BP1 phosphorylation in cell lines, with similar biological effects seen in some primary human lymphoblast samples. These data show that TSLP can promote survival of pre-B ALL cells and antagonize the effects of MTIs. These findings suggest that IL-7Ralpha chain is responsible for transducing the survival signal that overcomes MTI-mediated growth inhibition in pre-B ALL. Thus, further exploration of the IL-7Ralpha pathway may identify potential therapeutic targets in the treatment of ALL. Our data illustrate that growth-factor-mediated signaling may provide one mechanism of MTI resistance.

CD4+ T Cell-Specific Deletion of IL-4 Receptor α Prevents Ovalbumin-Induced Anaphylaxis by an IFN-γ-Dependent Mechanism

IL-4Ralpha-mediated STAT6 activation serves an essential role in various animal models of allergy and asthma at both the sensitization and effector phases. IL-4 and IL-13 signaling via the IL-4Ralpha chain exacerbates murine anaphylaxis, but the cell-specific requirements for IL-4Ralpha expression are unclear. The purpose of this study was to elucidate the mechanisms of systemic anaphylaxis to OVA in gene-targeted mice with a deletion of the IL-4Ralpha chain in the macrophage/neutrophil or CD4+ T lymphocyte population. Results demonstrated that anaphylaxis in this model was entirely dependent upon the FcgammaRII/III and was associated with mast cell degranulation. Expression of the IL-4Ralpha on CD4+ T cells, but not macrophages or neutrophils, was critical for severe anaphylaxis, characterized by diarrhea, hypothermia, and death. Ab depletion experiments demonstrated that IFN-gamma protected against mortality and severe intestinal pathology despite the presence of Ag and specific Ab. This protection was associated with reduced levels of mast cell protease, a marker of mast cell degranulation, suggesting that IFN-gamma may inhibit mast cell degranulation in vivo. These data suggest that it may be possible to limit the severity of anaphylaxis using rational therapies designed to increase numbers of IFN-gamma-producing cells by targeting IL-4Ralpha signaling in CD4+ T lymphocytes.

Induction of the IL-7 receptor α chain in mouse peripheral B cells by glucocorticoids

Expression of the IL-7Rα chain (IL-7Rα) is strictly regulated during development and maturation of lymphocytes. While T cells express the IL-7Rα in the periphery, B cells do not. Glucocorticoids (GCs) have pleiotypic effects on development and function of lymphocytes. Although GCs induce the transcription of IL-7Rα gene in T cells, their effect on B cells is largely unknown. Here, we show that GCs induce the transcription and expression of IL-7Rα in mouse peripheral B cells. This effect does not require de novo protein synthesis, because a protein synthesis inhibitor, cycloheximide, does not block the transcription. IL-7R signal pathway is intact in peripheral B cells because Stat5, one of the signal molecules of the IL-7Rα, is phosphorylated by IL-7 stimulation. We also observed that IL-7 simulation induces the transcription of Cis-1, one of the target genes of Stat5. Furthermore, GC-induced IL-7Rα can transmit survival signal in B cells. Therefore, this study demonstrates that GCs induce the transcription and expression of functional IL-7Rα in peripheral B cells, and implies a potential role of the IL-7R in survival of B cells.

Soluble Interleukin (IL)-15Rα Is Generated by Alternative Splicing or Proteolytic Cleavage and Forms Functional Complexes with IL-15

Interleukin 15 (IL-15) is a pleiotropic cytokine that is hardly detectable in biological fluids. Here, we show that IL-15 forms functional heterocomplexes with soluble high affinity IL-15 receptor alpha (IL-15Ralpha) chain in mouse serum and cell-conditioned medium, which prevents IL-15 detection by ELISA. We also demonstrate that two soluble IL-15Ralpha (sIL-15Ralpha) sushi domain isoforms are generated through a novel alternative splicing mechanism within the IL-15Ralpha gene. These isoforms potentiate IL-15 action by promoting the IL-15-mediated proliferation of the CTLL cell line and interferon gamma production by murine NK cells, which suggests a role in IL-15 transpresentation. Conversely, a full-length sIL-15Ralpha ectodomain released by tumor necrosis factor-alpha-converting enzyme (TACE)-dependent proteolysis inhibits IL-15 activity. Thus, a dual mechanism of sIL-15Ralpha generation exists in mice, giving rise to polypeptides with distinct properties, which regulate IL-15 function.

Interdependency of MHC Class II/Self-Peptide and CD1d/Self-Glycolipid Presentation by TNF-Matured Dendritic Cells for Protection from Autoimmunity

Dendritic cells (DC) are key regulators of T cell immunity and tolerance. NKT cells are well-known enhancers of Th differentiation and regulatory T cell function. However, the nature of the DC directing T and NKT cell activation and polarization as well as the role of the respective CD1d Ags presented is still unclear. In this study, we show that peptide-specific CD4(+)IL-10(+) T cell-mediated full experimental autoimmune encephalomyelitis (EAE) protection by TNF-treated semimatured DCs was dependent on NKT cells recognizing an endogenous CD1d ligand. NKT cell activation by TNF-matured DCs induced high serum levels of IL-4 and IL-13 which are absent in NKT cell-deficient mice, whereas LPS plus anti-CD40-treated fully mature DCs induce serum IFN-gamma. In the absence of IL-4Ralpha chain signaling or NKT cells, no complete EAE protection was achieved by TNF-DCs, whereas transfer of NKT cells into Jalpha281(-/-) mice restored it. However, activation of NKT cells alone was not sufficient for EAE protection and early serum Th2 deviation. Simultaneous activation of NKT cells and CD4(+) T cells by the same DC was required for EAE protection. Blocking experiments demonstrated that NKT cells recognize an endogenous glycolipid presented on CD1d on the injected DC. Together, this indicates that concomitant and interdependent presentation of MHC II/self-peptide and CD1d/self-isoglobotrihexosylceramide to T and NKT cells by the same partially or fully matured DC determines protective and nonprotective immune responses in EAE.

Nippostrongylus brasiliensis: Identification of intelectin-1 and -2 as Stat6-dependent genes expressed in lung and intestine during infection

Elimination of the helminth parasite Nippostrongylus brasiliensis from infected mice is mediated by IL-4 or IL-13 and dependent on the IL-4Rα chain and the transcription factor Stat6 in non-hematopoietic cells. However, it is not clear which Stat6-dependent effector molecules mediate worm expulsion. We identified intelectin-1 and -2 as Stat6-dependent genes that are induced during infection. Intelectins can bind galactofuranose, a sugar present only in microorganisms and might therefore serve as microbial pattern element. To analyze whether constitutive expression of intelectin-1 or -2 leads to accelerated pathogen clearance, transgenic mice were generated which express high levels of these genes selectively in the lung. Infection with N. brasiliensis or Mycobacterium tuberculosis did not result in accelerated pathogen clearance in transgenic as compared to wild-type mice. Further, no significant modulation of the immune response in lung or lymph nodes was observed. Thus, under these conditions, intelectins did not enhance pathogen clearance.

Molecular cloning and characterization of Duck CD25

The IL-2Rα chain (CD25, Tac) is an essential component of high affinity IL-2Rs, playing critical role for the immune specificity of antigen-activated T-cell clonal expansion. Up to now, no duck cytokine receptor has been described. Here, the cDNA segment of a duck cytokine receptor (duCD25), encoding a 226 aa precursor protein with a 20 aa signal peptide, was isolated. Then a novel mouse monoclonal antibody (mAb) was generated using the prokaryotically expressed duCD25 protein as immunogen. Using this mAb, the endogenous duCD25 molecule was localized on the surface of duck lymphocytes, and the duck IL-2-induced lymphocyte proliferation was further inhibited. Furthermore, flow cytometry analysis showed that duCD25 positive cells were upregulated in ducks infected with avian influenza virus (H9N2). Our findings confirm that duCD25 is a receptor of duck interleukin-2, and duCD25 positive cells play a potential role in H9N2 virus infection.