Nicotinamide-phosphoribosyl transferase (NAMPT) constitutes the rate-limiting enzyme in the NAD salvage pathway that is essential for the maintenance of cellular NAD levels. Upon inflammation including inflammatory bowel diseases (IBD), NAMPT is strongly induced and thereby impedes an increased cellular NAD-wastage promoted by NAD-depleting enzymes. These constitute an important mechanistic link between inflammatory, metabolic and transcriptional pathways and NAD-metabolism. Here, we investigated the efficacy of NAMPT inhibition by the small-molecule inhibitor FK866 in models of acute colitis, colitis-associated cancer and on lamina propria mononuclear cells (LPMNC) from IBD patients. FK866 mitigated colitis and suppressed inflammation-associated tumorigenesis. NAMPT blockade resulted in NAD-depletion that was associated with reduced abundances and activities of PARP1, Sirt6 and CD38, decreased inflammatory monocytes, macrophages and activated T-cells. FK866-treatment effectively supressed cytokine production in human LPMNC and was further associated with an altered monocyte/macrophage biology, skewing macrophages towards an anti-inflammatory M2 phenotype. Our data suggest that targeting the NAD immunometabolism by FK866 constitutes a promising therapeutic approach in acute intestinal inflammation and colitis-associated tumorigenesis.

Protein glycosylation plays an important role in protein stability, folding, and secretion but presents a major challenge for protein crystallization. Crystallization of highly glycosylated proteins is often difficult because of conformational and chemical inhomogeneity of the glycan decorations. Therefore, it is almost always necessary to modify the material to obtain a conformational homogenous protein that can crystallize. (1) We have explored several avenues in the case of the human 87 kDa glycoprotein afamin. Afamin (AFM), a member of the albumin gene family, is mainly expressed in the liver and secreted into the bloodstream. (2) Elevated afamin plasma concentrations are associated with major diseases such as metabolic syndrome and cancer;(3) however, pathophysiological functions are largely unknown. Therefore, we are pursuing the crystal structure of various forms of recombinantly expressed human afamin (rhAFM) for structure guided exploration and analysis of its function. Multiple variants of rhAFM are pursued: 1) fully glycosylated wild-type rhAFM (expressed in CHO cell lines); 2) rhAFM complexed with Fab fragments of two anti-AFM mAbs, 3) enzymatic deglycosylation of rhAFM with PGNaseF, 4) partially glycosylated rhAFM (expressed in glycosylation-deficient Lec1-CHO cells); and 5) glycosylation-free rhAFM, obtained from HEK293 cells virally transfected with a cDNA mutant lacking all 5 glycosylation sites by replacement of ASN with ASP. Yields of up to 2 mg/mL glycosilation free mutant rhAFM in HEK273 cells have been comparable to native rhAFM. C-terminal His6-tagged rhAFM was captured by Ni-IMAC from serum-free cell culture supernatants. All variants and Fab-AFM complexes were polished via SEC yielding pure products per SDS-PAGE and immunoblot analysis. Crystals have been obtained from Fab fragments and crystalline spherulites which are useful for microseeding from Fab-AFM complexes.

Dendritic cells (DCs) are potent antigen-presenting cells with a promising potential in cancer immunotherapy. Cbl proteins are E3 ubiquitin ligases and have been implicated in regulating the functional activity of various immune cells. As an example, c-Cbl negatively affects DC activation. We here describe that another member of the Cbl-protein family (i.e. Cbl-b) is highly expressed in murine bone-marrow-derived DCs (BMDCs). Differentiation of cblb-/- bone marrow mononuclear cells into classical BMDCs is unaltered, except enhanced induction of DEC-205 (CD205) expression. When tested in mixed-lymphocyte reaction (MLR), cblb-/- BMDCs exhibit increased allo-stimulatory capacity in vitro. BMDCs were next in vitro stimulated by various toll like receptor (TLR)-agonists (LPS, Poly(I:C), CpG) and exposed to FITC-labeled dextran. Upon TLR-stimulation, cblb-/- BMDCs produce higher levels of proinflammatory cytokines (IL-1α, IL-6 and TNF-α) and exhibit a slightly higher level of FITC-dextran uptake. To further characterize the functional significance of cblb-/- BMDCs we tested them in antigen-specific T cell responses against ovalbumin (OVA) protein and peptides, activating either CD8(+) OT-I or CD4(+) OT-II transgenic T cells. However, cblb-/- BMDCs are equally effective in inducing antigen-specific T cell responses when compared to wildtype BMDCs both in vitro and in vivo. The migratory capacity into lymph nodes during inflammation was similarly not affected by the absence of Cbl-b. In line with these observations, cblb-/- peptide-pulsed BMDCs are equally effective vaccines against OVA-expressing B16 tumors in vivo when compared to wildtype BMDCs. We conclude that in contrast to c-Cbl, Cbl-b plays only a limited role in the induction of Ag-specific T cell responses by murine BMDCs in vitro and in vivo.

Activation of NF- κ B (nuclear factor of kappa light chain gene enhancer in B cells) in response to DNA damage is considered to contribute to repair of genetic lesions, increased cell survival and cytokine release. The molecular mechanisms orchestrating this cytoplasmic event involve core components of the nuclear DNA damage response machinery, including ATM-kinase (ataxia telangiectasia mutated kinase) and PARP-1 (poly (ADP-ribose) polymerase 1). The physiological consequences of defective NF- κ B activation in this context, however, remain poorly investigated. Here we report on the role of the ‘p53-induced protein with a death domain’, PIDD, which appears rate limiting in this process, as is PARP-1. Despite impaired NF- κ B activation, DNA damage did not increase cell death or reduce clonal survival of various cell types lacking PIDD, such as mouse embryonic fibroblasts or stem and progenitor cells of the hematopoietic system. Furthermore, lymphomagenesis induced by γ -irradiation (IR) was unaffected by deficiency for PIDD or PARP-1, indicating that loss of DNA damage-triggered NF- κ B signalling does not affect IR-driven tumorigenesis. However, loss of either gene compromised cytokine release after acute IR injury. Hence, we propose that NF- κ B’s most notable function after DNA damage in primary cells is related to the release of cytokines, thereby contributing to sterile inflammation.

Abstract 957 Introduction: Various approaches to induce immunological rejection of tumors including transfer of autologous tumor infiltrating lymhocytes (TIL) after ex vivo clonal expansion or application of ex vivo transduced antigen specific T cell (TCR) transgenic T cells have been elaborated. In general, adoptive T cell transfer (ATC) has been combined with lympho-depleting agents (e.g. cyclophosphamide). However, the therapeutic efficacy of these cancer immunotherapy approaches is limited due to insufficient in vivo activation, expansion and survival of transferred effector immune cells, which is mainly due to suppressive mileu signals and immune evasion mechanisms induced by TGF-β. The E3 ubiquitin ligase Cbl-b is a key regulator of T cell activation and is assumed to confer TGF-β resistance. Thus we performed a proof-of-concept study evaluating Cbl-b targeting as “intracellular adjuvant” strategy to improve ATC for cancer immunotherapy. Material and Methods: We first tested the in vitro sensitivity of CTL towards TGF-β mediated immuno-suppressive cues and then in vivo evaluated the anti-tumor reactivity of cblb-deficient cytotoxic T lymphocytes (CTL) in murine tumor models alone or in combination with a dendritic cell (DC) vaccine. Results: Cblb-deficient CTL are hyper-responsive to TCR/CD28-stimulation in vitro and protected from the negative cues induced by TGF-β as determined by quantification fo IFN-g secretion and quantification of their proliferative capacity. Unexpectedly, adoptive transfer of polyclonal, non TCR-transgenic cblb-deficient CD8+ CTL, however, is not sufficient to reject B16ova or EG7 tumors in vivo, which is in clear contrast to previous reports using lymphopenic animals receiving adoptively transferred TCR-transgenic T cells. Thus, we next evaluated in vivo re-activation of adoptively transferred cblb-deficient T cells by a DC vaccine (i.e. SIINFEKL-pulsed DC). In strict contrast to ATC monotherapy, this approach now markedly delays tumor outgrowth and significantly increase survival rates, which is paralleled by an increased CTL infiltration rate to the tumor site and an enrichment of ova-specific and IFN-g-secreting CTL in the draining lymph nodes. Moreover, compared to wild-type CTL, cblb-deficient mice vaccinated with the DC vaccine show an increased cytolytic activity in vivo. Conclusions: In summary, we provide experimental evidence that genetic inactivation of cblb in polyclonal, non-TCR transgenic adoptively transferred CTL might serve as a novel “adjuvant approach”, suitable to augment the effectiveness of anti-cancer immunotherapies using ATC in immune-competent recipients. Disclosures: No relevant conflicts of interest to declare.

The main function of mature T cells is to recognize and respond to foreign antigens by a complex activation process involving differentiation of the resting cell to a proliferating lymphoblast actively secreting immunoregulatory lymphokines or displaying targeted cytotoxicity, ultimately leading to recruitment of other cell types and initiation of an effective immune response. In order to understand the physiology and pathophysiology of T lymphocytes, it is necessary to decode the biochemical processes that integrate signals from antigen, cytokine, integrin and death receptors. The principal upon which our work is based is to explore and identify gene products of distinct members of the AGC family of protein serine/threonine kinases as key players mediating cell growth regulation. Given the established important role of PKC theta as regulator of T cell fate and knowing that several other PKC isotypes are also expressed in T cells at a high level, we now summarize the physiological and non-redundant functions of PKC alpha, beta, delta, epsilon, zeta and theta isotypes in T cells. This review describes the current knowledge of the physiological and non-redundant functions of the PKC gene products in T cells.

Protein kinase C theta (PKC theta) is unique among PKC isozymes in its translocation to the center of the immune synapse in T cells and its unique downstream signaling. Here we show that the hematopoietic protein tyrosine phosphatase (HePTP) also accumulates in the immune synapse in a PKC theta-dependent manner upon antigen recognition by T cells and is phosphorylated by PKC theta at Ser-225, which is required for lipid raft translocation. Immune synapse translocation was completely absent in antigen-specific T cells from PKC theta(-/-) mice. In intact T cells, HePTP-S225A enhanced T-cell receptor (TCR)-induced NFAT/AP-1 transactivation, while the acidic substitution mutant was as efficient as wild-type HePTP. We conclude that HePTP is phosphorylated in the immune synapse by PKC theta and thereby targeted to lipid rafts to temper TCR signaling. This represents a novel mechanism for the active immune synapse recruitment and activation of a phosphatase in TCR signaling.

This international workshop on key signalling molecules in lymphocyte activation and immune regulation was held in Grossziethen, Germany from November 02-04, 2005 and brought together molecular, cellular, and clinical immunologists whose common goal is to develop ways of manipulating the immune response in order to avert T cell effector functions that are of significant relevance for pathogenesis in different diseases, including dermatological (psoriasis, atopic dermatitis and allergic contact allergy) and other indications (e.g. asthma, rheumatoid arthritis, multiple sclerosis and transplant rejection).

Using model tumor T cell lines, protein kinase C (PKC) (cid:1) has been implicated in IL-2 cytokine promoter activation in response to Ag receptor stimulation. In this study, for the first time, PKC (cid:1) null mutant mice are analyzed and display normal T and B lymphocyte development. Peripheral CD3 (cid:3) PKC (cid:1) -deficient T cells show unimpaired activation-induced IL-2 cytokine secretion, surface expression of CD25, CD44, and CD69, as well as transactivation of the critical transcription factors NF-AT, NF- (cid:4) B, AP-1, and STAT5 in vitro. Nevertheless, CD3/CD28 Ab- and MHC alloantigen-induced T cell proliferation and IFN- (cid:5) production are severely impaired in PKC (cid:1) (cid:2) / (cid:2) CD3 (cid:3) T cells. Consistently, PKC (cid:1) -deficient CD3 (cid:3) T cells from OVA-immunized PKC (cid:1) -deficient mice exhibit markedly reduced recall proliferation to OVA in in vitro cultures. In vivo, PKC (cid:1) -deficient mice give diminished OVA-specific IgG2a and IgG2b responses following OVA immunization experiments. In contrast, OVA-specific IgM and IgG1 responses and splenic PKC (cid:1) (cid:2) / (cid:2) B cell proliferation are unimpaired. Our genetic data, thus, define PKC (cid:1) as the physiological in PBS with 1% BSA), and incubated fo r 4 h at37°C. As positive control, anti-OVA (chicken, denatured; AntibodyShop) was used. Secondary Abs peroxidase-monoclonal rat anti-mouse IgG1, peroxidase-rat anti-mouse IgG2a, peroxidase-rat anti-mouse IgG2b, and peroxidase-monoclonal rat anti-mouse IgM (all from Zymed Laboratories) were incubated at 4°C overnight and subsequently peroxidase signaling was detected by the addition of ABTS (Fluka BioChemika).

Correction to: Cell Death and Differentiation 2006; doi:10.1038/sj.cdd.4402000. Advance online publication, 16 June 2006 In this article, published online 16.06.06, the authors have identified there has been an error in the initial of one of the authors names. N Harald should have been H Niederegger.

PKCɛ has been strongly linked to cell activation and proliferation in many cell types, including leukemic T-cell lines. In particularly, an essential role of PKCɛ has been established in the IKK-β/I-κB/NF-κB transactivation cascade. To study the physiological function of PKCɛ in primary T-cells, we used our newly established PKCɛ null mice. Unexpectedly, however, we did not reveal any defect in the development and function of CD3+ T-cells. Proliferative responses as well as IL-2 cytokine secretion of PKCɛ-deficient T-cells induced by allogenic MHC, plate-bound anti-CD3 antibodies (with or without anti-CD28 costimulation), or mitogenic stimuli such as phorbol ester and Ca2+ ionophore were comparable with wild-type controls. Consistently, after CD3/CD28 engagement, deficiency of PKCɛ did not impair NF-κB transactivation as well as CD25, CD44 and CD69 induction. Thus, PKCɛ-deficient T-cells had similar physiological thresholds for activation in vitro. This finding suggests that PKCɛ plays a redundant role in TCR-induced regulation of T-cell proliferation.

Phosphopeptide mapping identified a major autophosphorylation site, phospho (p)Thr‐219, between the tandem C1 domains of the regulatory fragment in protein kinase C (PKC)θ. Confirmation of this identification was derived using (p)Thr‐219 antisera that reacted with endogenous PKCθ in primary CD3+ T cells after stimulation with phorbol ester, anti‐CD3 or vanadate. The T219A mutation abrogated the capacity of PKCθ to mediate NF‐κB, NF‐AT and interleukin‐2 promoter transactivation, and reduced PKCθ's ability in Jurkat T cells to phosphorylate endogenous cellular substrates. In particular, the T219A mutation impaired crosstalk of PKCθ with Akt/PKBα in NF‐κB activation. Yet, this novel (p)Thr‐219 site did not affect catalytic activity or second‐messenger lipid‐binding activity in vitro. Instead, the T219A mutation prevented proper recruitment of PKCθ in activated T cells. The PKCθT219A mutant defects were largely rescued by addition of a myristoylation signal to force its proper membrane localization. We conclude that autophosphorylation of PKCθ at Thr‐219 plays an important role in the correct targeting and cellular function of PKCθ upon antigen receptor ligation.

The family of protein kinases C (PKCs) has been implicated in signal transmission leading to apoptosis induction and/or survival. These effects are cell type and tissue dependent. Numerous studies employing phorbol ester, a pleiotropic PKC activator, strongly implicated PKC in apoptosis induction of thymocytes. However, phorbol esters activate both, the conventional PKCs (PKCα, β, γ) as well as the novel PKCs (δ, e, η and θ), the PKC isotype(s) selectively involved in this process have not been established. In this study we used selective pharmacological PKC inhibitors and our established set of PKC knockout mice to define the PKC isotype that is involved in cell death induction of thymocytes. Pharmacological inhibition of nPKCs and in particular gene ablation of PKCδ, results in a profound reduction of p53-dependent as well as independent apoptosis induction. In strict contrast, loss of conventional PKCs as well as loss of two other thymocyte-expressed nPKC family members, PKCe and PKCθ, does not significantly affect thymocyte apoptosis. Taken together, we define an essential and non-redundant pro-apoptotic role of PKCδ in regulating distinct signaling mechanisms that are required to provoke apoptosis of mouse double positive thymocytes in vitro.

Hydrophobic vitamins are transported in human plasma and extravascular fluids by carrier proteins. No specific protein has been described so far for vitamin E, which plays a crucial role in protecting against oxidative damage and disease. We report here the purification of a 75-kDa glycoprotein with vitamin E-binding properties by stepwise chromatography of lipoprotein-depleted human plasma and monitoring of vitamin E (alpha-tocopherol)-binding activity. Partial sequencing identified this protein as afamin, a previously described member of the albumin gene family with four or five potential N-glycosylation sites. Glycosylation analysis indicated that > 90% of the glycans were sialylated biantennary complex structures. The vitamin E-binding properties were confirmed using recombinantly expressed afamin. Qualitative and quantitative analysis of plasma and extravascular fluids revealed an abundant presence of this protein not only in plasma (59.8 +/- 13.3 mu g/mL) but also in extravascular fluids such as follicular (34.4 +/- 12.7 mu g/mL) and cerebrospinal (0.28 +/- 0.16 mu g/mL) fluids, suggesting potential roles for afamin in fertility and neuroprotection. Afamin is partly (13%) bound to plasma lipoproteins. Afamin and vitamin E concentrations significantly correlate in follicular and cerebrospinal fluids but not in plasma. The vitamin E association of afamin in follicular fluid was directly demonstrated by gel filtration chromatography and immunoprecipitation which complements the in vitro findings for purified native and recombinant afamin.