CD7 delivers a pro-apoptotic signal during galectin-1-induced T cell death

Galectin-1, an endogenous lectin expressed in lymphoid organs and immune-privileged sites, induces death of human and murine thymocytes and T cells. Galectin-1 binds to several glycoproteins on the T cell surface, including CD7. However, the T cell surface glycoprotein receptors responsible for delivering the galectin-1 death signal have not been identified. We show that CD7 is required for galectin-1-mediated death. This demonstrates a novel function for CD7 as a death trigger and identifies galectin-1/CD7 as a new biologic death signaling pair.     

Interleukin-7 links T lymphocyte and intestinal epithelial cell homeostasis

Interleukin-7 (IL-7) is a major survival factor for mature T cells. Therefore, the degree of IL-7 availability determines the size of the peripheral T cell pool and regulates T cell homeostasis. Here we provide evidence that IL-7 also regulates the homeostasis of intestinal epithelial cells (IEC), colon function and the composition of the commensal microflora. In the colon of T cell-deficient, lymphopenic mice, IL-7-producing IEC accumulate. IEC hyperplasia can be blocked by IL-7-consuming T cells or the inactivation of the IL-7/IL-7R signaling pathway. However, the blockade of the IL-7/IL-7R signaling pathway renders T cell-deficient mice more sensitive to chemically-induced IEC damage and subsequent colitis. In summary, our data demonstrate that IL-7 promotes IEC hyperplasia under lymphopenic conditions. Under non-lymphopenic conditions, however, T cells consume IL-7 thereby limiting IEC expansion and survival. Hence, the degree of IL-7 availability regulates both, T cell and IEC homeostasis.     

Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival

Phosphorylation of BAD, a pro-apoptotic member of the Bcl-2 protein family, on either Ser112 or Ser136 is thought to be necessary and sufficient for growth factors to promote cell survival. Here we report that Ser155, a site phosphorylated by protein kinase A (PKA), also contributes to cell survival. Ser112 is thought to be the critical PKA target, but we found that BAD fusion proteins containing Ala at Ser112 (S112A) or Ser136 (S136A) or at both positions (S112/136A) were still heavily phosphorylated by PKA in an in vitro kinase assay. BAD became insensitive to phosphorylation by PKA only when both Ser112 and Ser136, or all three serines (S112/136/155) were mutated to alanine. In HEK293 cells, BAD fusion proteins mutated at Ser155 were refractory to phosphorylation induced by elevation of cyclic AMP(cAMP) levels. Phosphorylation of the S112/136A mutant was >90% inhibited by H89, a PKA inhibitor. The S155A mutant induced more apoptosis than the wild-type protein in serum-maintained CHO-K1 cells, and apoptosis induced by the S112/136A mutant was potentiated by serum withdrawal. These data suggest that Ser155 is a major site of phosphorylation by PKA and serum-induced kinases. Like Ser112 and Ser136, phosphorylation of Ser155 contributes to the cancellation of the pro-apoptotic function of BAD.     

Interleukin-9 and T cell subsets

Comment on: Nowak EC, et al. J Exp Med 2009; 206:1653-60.     

Pim-1 kinase promotes inactivation of the pro-apoptotic Bad protein by phosphorylating it on the Ser112 gatekeeper site

Constitutive expression of the Pim-1 kinase prolongs survival of cytokine-deprived FDCP1 cells, partly via maintenance of Bcl-2 expression. Here, we show that Pim-1 colocalizes and physically interacts with the pro-apoptotic Bad protein and phosphorylates it in vitro on serine 112, which is a gatekeeper site for its inactivation. Furthermore, wild-type Pim-1, but not a kinase-deficient mutant, enhances phosphorylation of this site in FDCP1 cells and protects cells from the pro-apoptotic effects of Bad. Our results suggest that phosphorylation of Bad by Pim-1 is one of several mechanisms via which the Pim-1 kinase can enhance Bcl-2 activity and promote cell survival.     

Cortisol safeguards oogenesis by promoting follicular cell survival

Science China Life Sciences - The role of glucocorticoids in oogenesis remains to be elucidated. cyp11c1 encodes the key enzyme involved in the synthesis of cortisol, the major glucocorticoid in...     

Med1 controls CD8 T cell maintenance through IL-7R-mediated cell survival signalling

Under steady-state conditions, the pool size of peripheral CD8⁺ T cells is maintained through turnover and survival. Beyond TCR and IL-7R signals, the underlying mechanisms are less well understood. In the present study, we found a significant reduction of CD8⁺ T cell proportion in spleens but not in thymi of mice with T cell-specific deletion of Mediator Subunit 1 (Med1). A competitive transfer of wild-type (WT) and Med1-deficient CD8⁺ T cells reproduced the phenotype in the same recipients and confirmed intrinsic role of Med1. Furthermore, we observed a comparable degree of migration and proliferation but a significant increase of cell death in Med1-deficient CD8⁺ T cells compared with WT counterparts. Finally, Med1-deficient CD8⁺ T cells exhibited a decreased expression of interleukin-7 receptor α (IL-7Rα), down-regulation of phosphorylated-STAT5 (pSTAT5) and Bim up-regulation. Collectively, our study reveals a novel role of Med1 in the maintenance of CD8⁺ T cells through IL-7Rα/STAT5 pathway-mediated cell survival.     

Quantitative interplay between activating and pro-apoptotic signals dictates T cell responses

Antigen-presenting cells (APC) can express surface ligands with both T cell activating and inhibitory capacities, prompting the question of how responding T cells integrate opposing trans signals concurrently delivered by APC. To address this question in a quantitative fashion, we turned to protein transfer as a unique experimental approach that is well-suited for addressing such questions from a quantitative standpoint. Costimulatory (either B7-1*Fc(gamma1) or Fc(gamma1)*4-1BBL) and pro-apoptotic (Fc(gamma1)*FasL) Fc fusion proteins were quantitatively "painted" in varying ratios onto surrogate APC pre-coated with palmitated-protein A, the latter serving as a surface anchor. Evaluating the signaling potential of these various painted cells in a standard in vitro T cell proliferation assay, we demonstrated that at a given level of TCR triggering, the quantitative balance between costimulator (B7-1 or 4-1BBL) and FasL dictates the magnitude of the proliferative T cell response. Furthermore, when the costimulator density is kept constant, there is also a quantitative balance between TCR-directed and FasL signals. Interesting species-specific nai;ve versus memory T cell subset differences emerged with regard to susceptibility to Fas-mediated apoptosis and costimulator:FasL opposition. Taken together, these data demonstrate for the first time a quantitative interplay between activating and pro-apoptotic trans signals that dictates the magnitude of T cell responses.     

Microparticle delivery of Interleukin-7 to boost T-cell proliferation and survival

In HIV infections, homoeostasis of T cells is dysregulated such that there is a depletion of CD4⁺ T cells and a progressive loss of naïve CD4⁺ and CD8⁺ T cells. Methodologies that can improve the function of some or all of these cells will likely enhance immune responsiveness in HIV infection. Interleukin‐7 (IL‐7) is a cytokine that has been shown to be critical in homeostatic expansion of naïve CD8⁺ and CD4⁺ cells in lymphopenic hosts, as well as regulating effector T cell to memory T‐cell transition and memory T‐cell homeostasis. In animal studies and clinical trials, repeated injections of IL‐7 are used to boost both CD4⁺ and CD8⁺ cell counts. Daily injections, however, are painful, inconvenient, and provide a frequent route for pathogen entry. We developed a poly (D ,L ‐lactide‐co‐glycolide; PLGA) microparticle controlled release system to administer IL‐7 in which a single injection of microparticles can provide therapeutic delivery of IL‐7. IL‐7 encapsulated PLGA microparticles were first synthesized using a water/organic/water double emulsion method, release from the particles was then optimized using in vitro release studies and therapeutic effectiveness was finally studied in animal studies. These PLGA microparticles showed effective delivery of IL‐7 for 1 week in vitro. These results were translated to in vivo delivery as well, which was followed for 9 days. Controlled release of IL‐7 in mice demonstrated biological activity in both CD4⁺ and CD8⁺ T cells in mice, which was consistent with previously reported results using daily injections. Biotechnol. Bioeng. 2012; 109:1835–1843. © 2012 Wiley Periodicals, Inc.     

Cytokine-induced protein kinase B activation and Bad phosphorylation do not correlate with cell survival of hemopoietic cells.

Activation of phosphoinositide-3 kinases (PI3Ks), their downstream target protein kinase B (PKB), and phosphorylation of Bad have all been implicated in survival signaling in many systems. However, it is not known whether these events are sufficient or necessary to universally prevent apoptosis. To address this issue, we have used three different factor-dependent hemopoietic cell lines, MC/9, BaF/3, and factor-dependent (FD)-6, which respond to a range of cytokines, to investigate the relationship between PI3K, PKB, and Bad activity with survival. The cytokines IL-3, IL-4, stem cell factor (SCF), GM-CSF, and insulin all induced the rapid and transient activation of PKB in responsive cell lines. In all cases, cytokine-induced PKB activation was sensitive to inhibition by the PI3K inhibitor, LY294002. However, dual phosphorylation of the proapoptotic protein Bad was found not to correlate with PKB activation. In addition, we observed cell-type-specific differences in the ability of the same cytokine to induce Bad phosphorylation. Whereas IL-4 induced low levels of dual phosphorylation of Bad in FD-6, it was unable to in MC/9 or BaF/3. Insulin, which was the most potent inducer of PKB in FD-6, induced barely detectable Bad phosphorylation. In addition, the ability of a particular cytokine to induce PKB activity did not correlate with its ability to promote cell survival and/or proliferation. These data demonstrate that, in hemopoietic cells, activation of PKB does not automatically confer a survival signal or result in phosphorylation of Bad, implying that other survival pathways must be involved.     

Adhesion and degranulation-promoting adapter protein (ADAP) positively regulates T cell sensitivity to antigen and T cell survival

The hemopoietic specific adapter protein ADAP (adhesion and degranulation-promoting adapter protein) positively regulates TCR-dependent, integrin-mediated adhesion and participates in signaling pathways downstream of the TCR that result in T cell activation. The specific role of ADAP in regulating Ag-dependent T cell interactions with APCs and T cell activation following Ag stimulation is not known. We used ADAP-/- DO11.10 T cells to demonstrate that ADAP promotes T cell conjugation to Ag-laden APCs. Complementary in vitro and in vivo approaches reveal that ADAP controls optimal T cell proliferation, cytokine production, and expression of the prosurvival protein Bcl-xL in response to limiting Ag doses. Furthermore, ADAP is critical for clonal expansion in vivo independent of Ag concentration under conditions of low clonal abundance. These results suggest that ADAP regulates T cell activation by promoting Ag-dependent T cell-APC interactions, resulting in enhanced T cell sensitivity to Ag, and by participating in prosurvival signaling pathways initiated by Ag stimulation.     

Mutual antagonism of TGF-beta and Interleukin-2 in cell survival and lineage commitment of induced regulatory T cells

Transforming growth factor beta (TGF-β)- and Interleukin-2 (IL-2)-mediated signaling enables the generation and expansion of induced regulatory T (iTreg) cells that carry high hopes for the treatment of chronic inflammatory and autoimmune diseases. Knowledge about factors stabilizing their lineage commitment and lifespan, however, is limited. Here, we investigated the behavior of iTreg cells, derived from apoptosis-defective mouse mutants, during activated cell autonomous cell death, triggered by cytokine-deprivation, or activation-induced cell death (AICD) after restimulation of the T-cell receptor, and compared these responses with those of effector T cells. We observed that iTreg cells were much more sensitive to IL-2-deprivation but poorly susceptible to AICD. In fact, when apoptosis was compromised, T-cell receptor (TCR)-religation resulted in methylation-independent, ERK- and PI3K/mTOR-mediated loss of Foxp3 expression, impaired suppressive capacity and effector cytokine production. Although iTreg cells prevented colitis induction they rapidly lost Foxp3-GFP expression and gained ability to produce effector cytokines thereby imposing Th1 cell fate on resident effector cells. Surprisingly, iTreg cell conversion itself was limited by TGF-β-mediated Bim/Bcl2L11-dependent apoptosis. Hence, the very same cytokine that drives the generation of iTreg cells can trigger their demise. Our results provide novel insights in iTreg cell biology that will assist optimization of iTreg-based therapy.     

The downregulation of the pro-apoptotic protein Par-4 is critical for Ras-induced survival and tumor progression

Inhibition of apoptosis is an important characteristic of oncogenic transformation. The Par-4 gene product has recently been shown to be upregulated in cells undergoing apoptotic cell death, and its ectopic expression was shown to be critical in apoptosis. We demonstrate that expression of oncogenic Ras promotes a potent reduction of Par-4 protein and mRNA levels through a MEK-dependent pathway. In addition, the expression of permanently active mutants of MEK, Raf-1 or zetaprotein kinase C but not of phosphatidylinositol 3-kinase (PI 3-kinase) is sufficient to decrease Par-4 levels. These effects are independent of p53, p16 and p19, and were detected not only in fibroblast primary cultures but also in NIH 3T3 and HeLa cells, indicating that they are not secondary to Ras actions on cell cycle regulation. Importantly, restoration of Par-4 levels to normal in Ras-transformed cells makes these cells sensitive to the pro-apoptotic actions of tumor necrosis factor-alpha under conditions in which PI 3-kinase is inhibited and also severely impairs colony formation in soft agar and tumor development in nude mice, as well as increases the sensitivity of these tumors to camptothecin. This indicates that the downregulation of Par-4 by oncogenic Ras is a critical event in tumor progression.     

Interleukin-7 and Toll-Like Receptor 7 Induce Synergistic B Cell and T Cell Activation

Objectives

To investigate the potential synergy of IL-7-driven T cell-dependent and TLR7-mediated B cell activation and to assess the additive effects of monocyte/macrophages in this respect.

Methods

Isolated CD19 B cells and CD4 T cells from healthy donors were co-cultured with TLR7 agonist (TLR7A, Gardiquimod), IL-7, or their combination with or without CD14 monocytes/macrophages (T/B/mono; 1 : 1 : 0,1). Proliferation was measured using ³H-thymidine incorporation and Ki67 expression. Activation marker (CD19, HLA-DR, CD25) expression was measured by FACS analysis. Immunoglobulins were measured by ELISA and release of cytokines was measured by Luminex assay.

Results

TLR7-induced B cell activation was not associated with T cell activation. IL-7-induced T cell activation alone and together with TLR7A synergistically increased numbers of both proliferating (Ki67⁺) B cells and T cells, which was further increased in the presence of monocytes/macrophages. This was associated by up regulation of activation markers on B cells and T cells. Additive or synergistic induction of production of immunoglobulins by TLR7 and IL-7 was associated by synergistic induction of T cell cytokines (IFNγ, IL-17A, IL-22), which was only evident in the presence of monocytes/macrophages.

Conclusions

IL-7-induced CD4 T cell activation and TLR7-induced B cell activation synergistically induce T helper cell cytokine and B cell immunoglobulin production, which is critically dependent on monocytes/macrophages. Our results indicate that previously described increased expression of IL-7 and TLR7 together with increased numbers of macrophages at sites of inflammation in autoimmune diseases like RA and pSS significantly contributes to enhanced lymphocyte activation.     

Conformational changes and protein stability of the pro-apoptotic protein Bax

Pro-apoptotic Bax is a soluble and monomeric protein under normal physiological conditions. Upon its activation substantial structural rearrangements occur: The protein inserts into the mitochondrial outer membrane and forms higher molecular weight oligomers. Subsequently, the cells can undergo apoptosis. In our studies, we focused on the structural rearrangements of Bax during oligomerization and on the protein stability. Both protein conformations exhibit high stability against thermal denaturation, chemically induced unfolding and proteolytic processing. The oligomeric protein is stable up to 90 °C as well as in solutions of 8 M urea or 6 M guanidinium hydrochloride. Helix 9 appears accessible in the monomer but hidden in the oligomer assessed by proteolysis. Tryptophan fluorescence indicates that the environment of the C-terminal protein half becomes more apolar upon oligomerization, whereas the loop region between helices 1 and 2 gets solvent exposed.     

Binding of the immunity protein inactivates colicin M

Colicin M (Cma) displays a unique mode of action in that it inhibits peptidoglycan and lipopolysaccharide biosynthesis through interference with bactoprenyl phosphate recycling. Protection of Cma-producing cells by the immunity protein (Cmi) was studied. The amount of Cmi determined the degree of inhibition of in vitro peptidoglycan synthesis by Cma. In cells, immunity breakdown could be achieved by overexpression of the Cma uptake system. Full immunity was restored after raising the cmi gene copy number. In sphaeroplasts, Cmi was degraded by trypsin, but this could be prevented by the addition of Cma. The N-terminal end includes the only hydrophobic amino acid sequence of Cmi, suggesting a function in anchoring of Cmi in the cytoplasmic membrane. It is proposed that Cmi does not act catalytically but binds Cma at the periplasmic face of the cytoplasmic membrane, thereby resulting in Cma inactivation. Two other possible modes of colicin M immunity, interference of Cmi with the uptake of Cma, and interaction of Cmi with the target of Cma, were ruled out by the data.