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Antibodies that recognize the ATP-gated P2X7 ion channel are etablished research tools. Nanobodies correspond to the antigen-binding variable immunoglobulin domain (VHH) of heavy chain antibodies that naturally occur in camelids. Nanobodies display better solubility than the variable domains (VH) of conventional antibodies. Therefore, it is much easier to construct bivalent and multivalent fusion proteins with nanobodies than with VH domains or with paired VH-VL domains. Moreover, nanobodies can bind functional crevices that are poorly accessbile to conventional VH-VL domains. This makes nanobodies particulary well suited as functional modulators. Here we provide protocols to raise antibodies and nanobodies against mouse and human P2X7 using cDNA-immunization. This approach evokes antibodies and nanobodies that recognize the P2X7 ion channel in native confirmation, some of which inhibit or potentiate gating of P2X7 by extracellular ATP. Furthermore, we developed protocols for producing P2X7-specific nanobodies and antibodies in vivo using rAAV vectors (AAVnano). This approach can be used either to durably inhibit or potentiate P2X7 function in vivo, or to deplete P2X7-expressing cells.

Liver transplant recipients (LTRs) show a decreased immune response after 2 severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) vaccinations compared with healthy controls (HCs). Here, we investigated the immunogenicity of additional vaccinations.In this prospective study, humoral (anti-SARS-CoV-2 receptor-binding domain [anti-S RBD]) and cellular (interferon-gamma release assay) immune responses were determined after mRNA-based SARS-CoV-2 vaccination in 106 LTRs after a third vaccination and in 36 LTRs after a fourth vaccination. Patients with anti-S RBD antibody levelsgt;0.8 arbitrary unit (AU)/mL after vaccination were defined as responders.After 3 vaccinations, 92% (97/106) of LTRs compared with 100% (28/28) of HCs were responders. However, the antibody titer of LTRs was lower compared with HCs (1891.0 vs 21,857.0 AU/mL; Plt; .001). Between a second and third vaccination (n = 75), the median antibody level increased 67-fold in LTRs. In patients seronegative after 2 vaccinations, a third dose induced seroconversion in 76% (19/25), whereas all HCs were already seropositive after 2 vaccinations. A spike-specific T-cell response was detected in 72% (28/39) after a third vaccination compared with 32% (11/34) after a second vaccination. Independent risk factors for a low antibody response (anti-S RBDlt;100 AU/mL) were first vaccination within the first year after liver transplant (odds ratio [OR], 8.00; P = .023), estimated glomular filtration ratelt;45 mL/min (OR, 4.72; P = .006), and low lymphocyte counts (OR, 5.02; P = .008). A fourth vaccination induced a 9-fold increase in the median antibody level and seroconversion in 60% (3/5) of previous non-responders.A third and fourth SARS-CoV-2 vaccination effectively increases the humoral and cellular immune response of LTRs, but to a lesser extent than in HCs. A fourth vaccination should be generally considered in LTRs.

CD38 is the major NAD+-hydrolyzing ecto-enzyme in most mammals. As a type II transmembrane protein, CD38 is also a promising target for the immunotherapy of multiple myeloma (MM). Nanobodies are single immunoglobulin variable domains from heavy chain antibodies that naturally occur in camelids. Using phage display technology, we isolated 13 mouse CD38-specific nanobodies from immunized llamas and produced these as recombinant chimeric mouse IgG2a heavy chain antibodies (hcAbs). Sequence analysis assigned these hcAbs to five distinct families that bind to three non-overlapping epitopes of CD38. Members of families 4 and 5 inhibit the GDPR-cyclase activity of CD38. Members of families 2, 4 and 5 effectively induce complement-dependent cytotoxicity against CD38-expressing tumor cell lines, while all families effectively induce antibody dependent cellular cytotoxicity. Our hcAbs present unique tools to assess cytotoxicity mechanisms of CD38-specific hcAbs in vivo against tumor cells and potential off-target effects on normal cells expressing CD38 in syngeneic mouse tumor models, i.e. in a fully immunocompetent background.

Murine T cells express the GPI-anchored ADP-ribosyltransferase 2.2 (ARTC2.2) on the cell surface. In response to T cell activation or extracellular NAD+ or ATP-mediated gating of the P2X7 ion channel ARTC2.2 is shed from the cell surface as a soluble enzyme. Shedding alters the target specificity of ARTC2.2 from cell surface proteins to secreted proteins. Here we demonstrate that shed ARTC2.2 potently ADP-ribosylates IFN-γ in addition to other cytokines. Using mass spectrometry, we identify arginine 128 as the target site of ADP-ribosylation. This residue has been implicated to play a key role in binding of IFN-γ to the interferon receptor 1 (IFNR1). Indeed, binding of IFN-γ to IFNR1 blocks ADP-ribosylation of IFN-γ. Moreover, ADP-ribosylation of IFN-γ inhibits the capacity of IFN-γ to induce STAT1 phosphorylation in macrophages and upregulation of the proteasomal subunit ß5i and the proteasomal activator PA28-α in podocytes. Our results show that ADP-ribosylation inhibits the signaling functions of IFN-γ and point to a new regulatory mechanism for controlling signaling by IFN-γ.

Rationale: CD38 is a target for the therapy of multiple myeloma (MM) with monoclonal antibodies such as daratumumab and isatuximab. Since MM patients exhibit a high rate of relapse, the development of new biologics targeting alternative CD38 epitopes is desirable. The discovery of single-domain antibodies (nanobodies) has opened the way for a new generation of antitumor therapeutics. We report the generation of nanobody-based humanized IgG1 heavy chain antibodies (hcAbs) with a high specificity and affinity that recognize three different and non-overlapping epitopes of CD38 and compare their cytotoxicity against CD38-expressing hematological cancer cells in vitro, ex vivo and in vivo. Methods: We generated three humanized hcAbs (WF211-hcAb, MU1067-hcAb, JK36-hcAb) that recognize three different non-overlapping epitopes (E1, E2, E3) of CD38 by fusion of llama-derived nanobodies to the hinge- and Fc-domains of human IgG1. WF211-hcAb shares the binding epitope E1 with daratumumab. We compared the capacity of these CD38-specific hcAbs and daratumumab to induce CDC and ADCC in CD38-expressing tumor cell lines in vitro and in patient MM cells ex vivo as well as effects on xenograft tumor growth and survival in vivo. Results: CD38-specific heavy chain antibodies (WF211-hcAb, MU1067-hcAb, JK36-hcAb) potently induced antibody-dependent cellular cytotoxicity (ADCC) in CD38-expressing tumor cell lines and in primary patient MM cells, but only little if any complement-dependent cytotoxicity (CDC). In vivo, CD38-specific heavy chain antibodies significantly reduced the growth of systemic lymphomas and prolonged survival of tumor bearing SCID mice. Conclusions: CD38-specific nanobody-based humanized IgG1 heavy chain antibodies mediate cytotoxicity against CD38-expressing hematological cancer cells in vitro, ex vivo and in vivo. These promising results of our study indicate that CD38-specific hcAbs warrant further clinical development as therapeutics for multiple myeloma and other hematological malignancies.

Extracellular adenine nucleotides participate in cell-to-cell communication and modulate the immune response. The concerted action of ecto-nucleotidases CD39 and CD73 plays a major role in the local production of anti-inflammatory adenosine, but both ecto-nucleotidases are rarely co-expressed by human T cells. The expression of CD39 on T cells increases upon T cell activation and is high at sites of inflammation. CD73, in contrast, disappears from the cellular membrane after activation. The possibility that CD73 could act in trans would resolve the conundrum of both enzymes being co-expressed for the degradation of ATP and the generation of adenosine. An enzymatically active soluble form of CD73 has been reported, and AMPase activity has been detected in body fluids of patients with inflammation and cancer. It is not yet clear how CD73, a glycosylphosphatidylinositol (GPI)-anchored protein, is released from the cell membrane, but plausible mechanisms include cleavage by metalloproteinases and shedding mediated by cell-associated phospholipases. Importantly, like many other GPI-anchored proteins, CD73 at the cell membrane is preferentially localized in detergent-resistant domains or lipid rafts, which often contribute to extracellular vesicles (EVs). Indeed, CD73-containing vesicles of different size and origin and with immunomodulatory function have been found in the tumor microenvironment. The occurrence of CD73 as non-cell-bound molecule widens the range of action of this enzyme at sites of inflammation. In this review, we will discuss the generation of non-cell-bound CD73 and its physiological role in inflammation.

Mouse T cells express the toxin-related ecto-ADP-ribosyltransferase ARTC2 that catalyzes the posttranslational ADP-ribosylation of cell surface proteins by transferring the ADP-ribose group of its substrate nicotinamide adenine dinucleotide (NAD

ATP and its metabolites are important extracellular signal transmitters acting on purinergic P2 and P1 receptors. Most cells can actively secrete ATP in response to a variety of external stimuli such as gating of the P2X7 receptor. We used Yac-1 murine lymphoma cells to study P2X7-mediated ATP release. These cells co-express P2X7 and ADP-ribosyltransferase ARTC2, permitting gating of P2X7 by NAD+-dependent ADP-ribosylation without the need to add exogenous ATP. Yac-1 cells released ATP into the extracellular space within minutes after stimulation with NAD+. This was blocked by pre-incubation with the inhibitory P2X7-specific nanobody 13A7. Gating of P2X7 for 3 h significantly decreased intracellular ATP levels in living cells, but these had returned to normal by 20 h. P2X7-mediated ATP release was dependent on a rise in cytosolic calcium and the depletion of intracellular potassium, but was not blocked by inhibitors of pannexins or connexins. We used genetically encoded FRET-based ATP sensors targeted to the cytosol to image P2X7-mediated changes in the distribution of ATP in 3T3 fibroblasts co-expressing P2X7 and ARTC2 and in Yac-1 cells. In response to NAD+, we observed a marked depletion of ATP in the cytosol. This study demonstrates the potential of ATP sensors as tools to study regulated ATP release by other cell types under other conditions. Electronic supplementary material The online version of this article (10.1007/s11302-019-09654-5) contains supplementary material, which is available to authorized users.

The ectoenzymes CD39 and CD73 degrade extracellular ATP to adenosine. ATP is released by stressed or damaged cells and provides pro-inflammatory signals to immune cells through P2 receptors. Adenosine, on the other hand, suppresses immune cells by stimulating P1 receptors. Thus, CD39 and CD73 can shape the quality of immune responses. Here we demonstrate that upregulation of CD39 is a consistent feature of activated conventional CD4+ and CD8+ T cells. Following stimulation in vitro, CD4+ and CD8+ T cells from human blood gained surface expression of CD39 but displayed only low levels of CD73. Activated human T cells from inflamed joints largely presented with a CD39+CD73- phenotype. In line, in spleens of mice with acute Listeria monocytogenes, listeria-specific CD4+ and CD8+ T cells acquired a CD39+CD73- phenotype. To test the function of CD39 in control of bacterial infection, CD39-deficient (CD39-/-) mice were infected with L. monocytogenes. CD39-/- mice showed better initial control of L. monocytogenes, which was associated with enhanced production of inflammatory cytokines. In the late stage of infection, CD39-/- mice accumulated more listeria-specific CD8+ T cells in the spleen than wildtype animals suggesting that CD39 attenuates the CD8+ T-cell response to infection. In conclusion, our results demonstrate that CD39 is upregulated on conventional CD4+ and CD8+ T cells at sites of acute infection and inflammation, and that CD39 dampens responses to bacterial infection.

Objective The CD4+ T cell immune response plays a pivotal role in the immunopathogenesis of human and experimental lupus nephritis, but the contribution of the Th17/interleukin-17 (IL-17) immune pathway to renal tissue injury in systemic lupus erythematosus (SLE) remains to be elucidated. The aim of this study was to characterize the function of the Th17/IL-17A immune response in 2 murine models of lupus nephritis. Methods IL-17A–deficient MRL/MPJ-Faslpr/2J (MRL/lpr) mice were generated, and the clinical course of nephritis was monitored by assessing the levels of albuminuria, extent of renal tissue injury, and functional parameters. In addition, lupus-prone (NZB × NZW)F1 (NZB/NZW) mice were treated with anti–IL-17A and anti–interferon-γ (anti-IFNγ) antibodies, and their effects on the clinical course of lupus nephritis were assessed. Results Characterization of renal IL-17A–producing and IFNγ-producing T cells in MRL/lpr and NZB/NZW mice revealed low numbers of infiltrating CD3+IL-17A+ cells. Renal IL-17A was mainly produced by CD4/CD8 double-negative CD3+ T cells and CD4+ Th17 cells. In contrast, the number of renal CD3+IFNγ+ cells continuously increased over time and largely consisted of typical CD4+ Th1 cells. IL-17A deficiency did not affect the morphologic or functional parameters in MRL/lpr mice with lupus nephritis, nor did IL-17A neutralization affect the clinical course of nephritis in NZB/NZW mice, but anti-IFNγ treatment attenuated the severity of the disease. Conclusion The Th17/IL-17A immune response plays no major role in the immunopathogenesis of lupus nephritis in MRL/lpr and NZB/NZW mice. Thus, the results of this study do not support the hypothesis that IL-17A targeting could be an intriguing new therapeutic approach for the management of proliferative lupus nephritis in SLE patients.

Mammalian ecto-ADP-ribosyltransferases (ecto-ARTs or also ARTCs) catalyze the ADP-ribosylation of cell surface proteins using extracellular nicotinamide adenine dinucleotide (NAD+) as substrate. By this post-translational protein modification, ecto-ARTs modulate the function of various target proteins. A functional role of ARTC2 has been demonstrated for peripheral immune cells such as T cells and macrophages. Yet, little is known about the role of ecto-ARTs in the central nervous system and on microglia. Here, we identified ARTC2.1 as the major ecto-ART expressed on murine microglia. ARTC2.1 expression was strongly upregulated on microglia upon co-stimulation with LPS and an ERK1/2 inhibitor or upon IFNβ stimulation. We identified several target proteins modified by ARTC2.1 on microglia with a recently developed mass spectrometry approach, including two receptors for immunoglobulin G (IgG), FcγR1 and FcγR2B. Both proteins were verified as targets of ARTC2.1 in vitro using a radiolabeling assay with 32P-NAD+ as substrate. Moreover, ADP-ribosylation of both targets strongly inhibited their capacity to bind IgG. In concordance, ARTC2.1 induction in WT microglia and subsequent cell surface ADP-ribosylation significantly reduced the phagocytosis of IgG-coated latex beads, which was unimpaired in NAD+/DTT treated microglia from ARTC2.1−/− mice. Hence, induction of ARTC2.1 expression under inflammatory conditions, and subsequent ADP-ribosylation of cell surface target proteins could represent a hitherto unnoticed mechanism to regulate the immune response of murine microglia.

The cell surface ecto-enzyme CD38 is a promising target antigen for the treatment of hematological malignancies, as illustrated by the recent approval of daratumumab for the treatment of multiple myeloma. Our aim was to evaluate the potential of CD38-specific nanobodies as novel diagnostics for hematological malignancies. We successfully identified 22 CD38-specific nanobody families using phage display technology from immunized llamas. Crossblockade analyses and in-tandem epitope binning revealed that the nanobodies recognize three different non-overlapping epitopes, with four nanobody families binding complementary to daratumumab. Three nanobody families inhibit the enzymatic activity of CD38 in vitro, while two others were found to act as enhancers. In vivo, fluorochrome-conjugated CD38 nanobodies efficiently reach CD38 expressing tumors in a rodent model within 2 hours after intravenous injection, thereby allowing for convenient same day in vivo tumor imaging. These nanobodies represent highly specific tools for modulating the enzymatic activity of CD38 and for diagnostic monitoring CD38-expressing tumors.

Leukocyte infiltration is a characteristic feature of human and experimental lupus nephritis and is closely correlated with loss of renal function. The chemokine receptor CCR5 is expressed on monocyte and T cell subsets and is thought to play an important role in recruiting these cells into inflamed organs. To investigate the functional role of CCR5 in lupus nephritis, CCR5-deficient mice were backcrossed onto the lupus-prone MRL- Faslpr(MRL/lpr) genetic background. Unexpectedly, CCR5−/−MRL/lpr mice developed an aggravated course of lupus nephritis in terms of glomerular tissue injury and albuminuria. Deterioration of the nephritis was associated with an overall increase in mononuclear cell infiltration into the kidney, whereas renal leukocyte subtype balance, systemic T cell response, and autoantibody formation were unaffected by CCR5 deficiency. Renal and systemic protein levels of the CCR5 ligand CCL3, which can also attract leukocytes via its alternate receptor CCR1, were significantly increased in nephritic CCR5−/−MRL/lpr mice. Further studies revealed that the systemic increase in the CCR5/CCR1 ligand is also observed in nonimmune CCR5−/−C57BL/6 mice and that this increase was due to a reduced clearance, rather than an overproduction, of CCL3. Taken together, our data support the hypothesis that CCR5-dependent consumption of its own ligands may act as a negative feedback loop to restrain local chemokine levels within inflamed tissues, thereby limiting inflammatory cell influx.

Mono-ADP-ribosylation of arginine is a protein modification in eukaryotic cells regulating protein activity and thereby influencing signal transduction and metabolism. Due to the complexity of the modification and the fragmentation pattern in MS/MS CID experiments, the identification of ADP-ribosylation sites in complex mixtures is difficult. Here we describe a two-step strategy, in the first step enriching and identifying potentially ADP-ribosylated proteins and in the second step identifying the sites of modification by a combination of LC/MS-, LC/MS(E) (MS at elevated fragmentation energy)- and LC/MS/MS experiments. Using this technique we could identify two ADP-ribosylation sites in TNFα digested with trypsin, protease V8 and both proteases and thereby demonstrate the specific ADP-ribosylation of TNFα. In complex samples the detection of ADP-ribosylated peptides requires further enrichment of the modified peptides. We tested various materials routinely used for the isolation of phosphopeptides. IMAC as well as TiO(2) chromatography were successfully applied for the selective enrichment of ADP-ribosylated model peptides.

T cells constitutively express low amounts of a toxin-related ADP-ribosylating ecto-enzyme, ART2.2. In inflammatory settings, cells release NAD, the substrate for ART2.2. The ART2.2 catalyzed ADP-ribosylation of cell surface proteins induces cell death. However, the low expression levels of ART2.2 have hampered analysis of ART2.2 in physiological settings. Here we report the generation of transgenic mice over-expressing ART2.2 under the control of the H2K promoter and Igμ enhancer. ART2.2 transgenic mice were healthy and fertile and exhibited normal development of the major lymphocyte subsets. Most T cells and a small subpopulation of B cells from transgenic mice showed more than 10-fold higher levels of ART2.2 expression than their wild-type counterparts. Exposure of ART2.2-transgenic T cells to low, submicromolar concentrations of NAD caused cell membrane alterations including uptake of propidium iodide, externalization of phosphatidylserine, and shedding of CD62L, while ART2.2-transgenic B cells were resistant to NAD. The ART2.2-overexpressing animals described here confirm that ART2.2 is an essential component for the regulation of T-cell functions by extracellular NAD and provide a useful tool to further elucidate the function of ART2.2 in vivo.

In addition to conventional antibodies, cartilaginous fish have evolved a distinctive type of immunoglobulin, designated as IgNAR, which lacks the light polypeptide chains and is composed entirely by heavy chains. IgNAR molecules can be manipulated by molecular engineering to produce the variable domain of a single heavy chain polypeptide (vNARs). These, together with the VHH camel domains, constitute the smallest naturally occurring domains able to recognize an antigen. Their special features, such as small size, long extended finger-like CDR3, and thermal and chemical stability, make them suitable candidates for biotechnological purposes. Here we describe the generation of two mouse monoclonal antibodies (MAbs), MAb 370-12 and MAb 533-10, that both specifically react with vNAR domains of the horn shark Heterodontus francisci. While the former recognizes a broad spectrum of recombinant vNAR proteins, the latter is more restricted. MAb 370-12 precipitated a single band from whole shark serum, which was identified as IgNAR by mass spectrometry. Additionally, we used MAb 370-12 to follow the IgNAR-mediated immune response of sharks during immunization protocols with two different antigens (complete cells and a synthethic peptide), thus corroborating that MAb 370-12 recognizes both isolated vNAR domains and whole IgNAR molecules. Both MAbs represent an affordable molecular, biochemical, and biotechnological tool in the field of shark single-domain antibodies.

Extracellular NAD+ affects the survival and function of regulatory T cells, and NAD-mediated depletion of regulatory T cells promotes anti-tumor responses in mice., CD4+CD25+FoxP3+ regulatory T cells (T reg cells) play a major role in the control of immune responses but the factors controlling their homeostasis and function remain poorly characterized. Nicotinamide adenine dinucleotide (NAD+) released during cell damage or inflammation results in ART2.2–mediated ADP-ribosylation of the cytolytic P2X7 receptor on T cells. We show that T reg cells express the ART2.2 enzyme and high levels of P2X7 and that T reg cells can be depleted by intravenous injection of NAD+. Moreover, lower T reg cell numbers are found in mice deficient for the NAD-hydrolase CD38 than in wild-type, P2X7-deficient, or ART2-deficient mice, indicating a role for extracellular NAD+ in T reg cell homeostasis. Even routine cell preparation leads to release of NAD+ in sufficient quantities to profoundly affect T reg cell viability, phenotype, and function. We demonstrate that T reg cells can be protected from the deleterious effects of NAD+ by an inhibitory ART2.2-specific single domain antibody. Furthermore, selective depletion of T reg cells by systemic administration of NAD+ can be used to promote an antitumor response in several mouse tumor models. Collectively, our data demonstrate that NAD+ influences survival, phenotype, and function of T reg cells and provide proof of principle that acting on the ART2–P2X7 pathway represents a new strategy to manipulate T reg cells in vivo.

ADP-ribosylation of host cell proteins is a common mode of cell intoxication by pathogenic bacterial toxins. Antibodies induced by immunization with inactivated ADP-ribosylating toxins provide efficient protection in case of some secreted toxins, e.g., diphtheria and pertussis toxins. However, other ADP-ribosylating toxins, such as Salmonella SpvB toxin, are secreted directly from the Salmonella-containing vacuole into the cytosol of target cells via the SPI-2 encoded bacterial type III secretion system, and thus are inaccessible to conventional antibodies. Small-molecule ADP-ribosylation inhibitors are fraught with potential side effects caused by inhibition of endogenous ADP-ribosyltransferases. Here, we report the development of a single-domain antibody from an immunized llama that blocks the capacity of SpvB to ADP-ribosylate actin at a molar ratio of 1:1. The single-domain antibody, when expressed as an intrabody, effectively protected cells from the cytotoxic activity of a translocation-competent chimeric C2IN-C/SpvB toxin. Transfected cells were also protected against cytoskeletal alterations induced by wild-type SpvB-expressing strains of Salmonella. This proof of principle paves the way for developing new antidotes against intracellular toxins Fil: Alzogaray, Vanina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Danquah, Welbeck. Hamburg University Medical Center; Alemania Fil: Aguirre, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Urrutia, Mariela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Berguer, Paula Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Garcia Vescovi, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Haag, Friedrich. Hamburg University Medical Center; Alemania Fil: Koch Nolte, Friedrich. Hamburg University Medical Center; Alemania Fil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Extracellular NAD induces the ATP-independent activation of the ionotropic P2X7 purinergic receptor (P2X7R) in murine T lymphocytes via a novel covalent pathway involving ADP-ribosylation of arginine residues on the P2X7R ectodomain. This modification is catalyzed by ART2.2, a GPI-anchored ADP-ribosyltransferase (ART) that is constitutively expressed in murine T cells. We previously reported that ART2.1, a related ecto-ART, is up-regulated in inflammatory murine macrophages that constitutively express P2X7R. Thus, we tested the hypothesis that extracellular NAD acts via ART2.1 to regulate P2X7R function in murine macrophages. Coexpression of the cloned murine P2X7R with ART2.1 or ART2.2 in HEK293 cells verified that P2X7R is an equivalent substrate for ADP-ribosylation by either ART2.1 or ART2.2. However, in contrast with T cells, the stimulation of macrophages or HEK293 cells with NAD alone did not activate the P2X7R. Rather, NAD potentiated ATP-dependent P2X7R activation as indicated by a left shift in the ATP dose-response relationship. Thus, extracellular NAD regulates the P2X7R in both macrophages and T cells but via distinct mechanisms. Although ADP-ribosylation is sufficient to gate a P2X7R channel opening in T cells, this P2X7R modification in macrophages does not gate the channel but decreases the threshold for gating in response to ATP binding. These findings indicate that extracellular NAD and ATP can act synergistically to regulate P2X7R signaling in murine macrophages and also suggest that the cellular context in which P2X7R signaling occurs differs between myeloid and lymphoid leukocytes.

The purinoreceptor P2X7 is expressed on subsets of T cells and mediates responses of these cells to extracellular nucleotides such as ATP or NAD+. We identified P2X7 as a molecule highly up-regulated on conventional CD8αβ+ and unconventional CD8αα+ T cells of the intestinal epithelium of mice. In contrast, CD8+ T cells derived from spleen, mesenteric lymph nodes, and liver expressed only marginal levels of P2X7. However, P2X7 was highly up-regulated on CD8+ T cells from spleen and lymph nodes when T cells were activated in the presence of retinoic acid. High P2X7 expression on intestinal CD8+ T cells as well as on CD8+ T cells incubated with retinoic acid resulted in enhanced sensitivity of cells to extracellular nucleotides. Both cell populations showed a high level of apoptosis following incubation with NAD+ and the ATP derivative 2′,3′-O-(benzoyl-4-benzoyl)-ATP, and injection of NAD+ caused selective in vivo depletion of intestinal CD8+ T cells. Following oral infection with Listeria monocytogenes, P2X7-deficient mice showed similar CD8+ T cell responses in the spleen, but enhanced responses in the intestinal mucosa, when compared with similarly treated wild-type control mice. Overall, our observations define P2X7 as a new regulatory element in the control of CD8+ T cell responses in the intestinal mucosa.

Mono ADP-ribosyltransferase 2 (ART2) is an ectoenzyme expressed on mouse T lymphocytes, which catalyze the transfer of ADP-ribose groups from NAD+ onto several target proteins. In vitro, ADP-ribosylation by ART2 activates the P2X7 ATP receptor and is responsible for NAD+-induced T cell death (NICD). Yet, the origin of extracellular NAD+ and the role of NICD in vivo remain elusive. In a model of acute inflammation induced by polyacrylamide beads, we demonstrate release of NAD+ into exudates during the early phase of the inflammatory response. This leads to T cell depletion in the draining lymph nodes from wild-type and, more severely, from mice lacking the CD38 NAD+ glycohydrolase, whereas no effect is observed in ART2-deficient animals. Intravenous injection of NAD+ used to exacerbate NICD in vivo results in fast and dramatic ART2- and P2X7-dependent depletion of CD4+ and CD8+ T lymphocytes, which can affect up to 80% of peripheral T cells in CD38−/− mice. This affects mainly naive T cells as most cells surviving in vivo NAD+ treatment exhibit the phenotype of recently activated/memory cells. Consistently, treatment with NAD+ abolishes primary Ab response to a T-dependent Ag in NICD-susceptible CD38−/− mice but has no effect on the secondary response when given several days after priming. Unexpectedly NAD+ treatment improves the response in their wild-type BALB/c counterparts. We propose that NAD+ released during early inflammation facilitates the expansion of primed T cells, through ART2-driven death of resting cells, thus contributing to the dynamic regulation of T cell homeostasis.

International audience; Ion channels are desirable therapeutic targets, yet ion channel-directed drugs with high selectivity and few side effects are still needed. Unlike small-molecule inhibitors, antibodies are highly selective for target antigens but mostly fail to antagonize ion channel functions. Nanobodies-small, single-domain antibody fragments-may overcome these problems. P2X7 is a ligand-gated ion channel that, upon sensing adenosine 5′-triphosphate released by damaged cells, initiates a proinflammatory signaling cascade, including release of cytokines, such as interleukin-1b (IL-1b). To further explore its function, we generated and characterized nanobodies against mouse P2X7 that effectively blocked (13A7) or potentiated (14D5) gating of the channel. Systemic injection of nanobody 13A7 in mice blocked P2X7 on T cells and macrophages in vivo and ameliorated experimental glomerulonephritis and allergic contact dermatitis. We also generated nanobody Dano1, which specifically inhibited human P2X7. In endotoxin-treated human blood, Dano1 was 1000 times more potent in preventing IL-1b release than small-molecule P2X7 antagonists currently in clinical development. Our results show that nanobody technology can generate potent, specific therapeu-tics against ion channels, confirm P2X7 as a therapeutic target for inflammatory disorders, and characterize a potent new drug candidate that targets P2X7.

Control of immunologic tolerance and homeostasis rely on Foxp3+CD4+CD25+ regulatory T cells (Tregs) that constitutively express the high affinity receptor for Interleukin-2, CD25. Tregs proliferate in response to injections of IL-2/anti-IL-2 antibody complexes or low doses of IL-2. However, little is known about endogenous mechanisms that regulate the sensitivity of CD25 to signaling by IL-2. Here we demonstrate that CD25 is ADP-ribosylated at Arg35 in the IL-2 binding site by ecto-ADP-ribosyltransferase ARTC2.2, a toxin-related GPI-anchored ecto-enzyme. ADP-ribosylation inhibits binding of IL-2 by CD25, IL-2- induced phosphorylation of STAT5 and IL-2-dependent cell proliferation. Our study elucidates an as-yet-unrecognized mechanism to tune IL-2 signaling. This newly found mechanism might thwart Tregs at sites of inflammation and thereby permit a more potent response of activated effector T cells.

Ubiquitously expressed CD38 and T cell-expressed ADP-ribosyltransferase 2 (ART2) are ectoenzymes competing for NAD substrate. CD38 exerts pleiotropic actions in hemopoietic and nonhemopoietic compartments via effects on calcium mobilization. ART2 is an ADP-ribosyltransferase on naive CD4+ and CD8+ T cells. ART2-catalyzed ADP-ribosylation of the P2X7 purinoreceptor elicits apoptosis. Transfer of a genetically disrupted CD38 allele into the autoimmune diabetes-prone NOD/Lt background accelerated diabetes onset in both sexes, whereas transfer of a disrupted ART2 complex had no effect. However, the fact that the accelerated pathogenesis mediated by CD38 deficiency required ART2 activity was demonstrated by combining both ART2 and CD38 deficiencies. Reciprocal bone marrow reconstitution studies demonstrated accelerated diabetes only when CD38-deficient bone marrow was transferred into CD38-deficient recipients. Neither decreases in β cell function nor viability were indicated. Rather, the balance between T-effectors and T-regulatory cells was disturbed in CD38-deficient but ART2-intact NOD mice. In these mice, significant reductions in total viable CD8+ T cells were observed. This was accompanied by an age-dependent increase in a diabetogenic CD8 clonotype. This in turn correlated with impaired T-regulatory development (10-fold reduction in Foxp3 mRNA expression). These changes were corrected when CD38 deficiency was combined with ART2 deficiency. Both ART2-deficient and CD38/ART2 combined deficient T cells were resistant to NAD-induced killing in vitro, whereas CD38-deficient but ART2-intact T cells showed increased sensitivity, particularly the CD4+CD25+ subset. Unexpectedly, diabetes development in the combined CD38/ART2 stock was strongly suppressed, possibly through epistatic interactions between genes linked to the targeted CD38 on Chromosome 5 and the ART2 complex on Chromosome 7.

ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD to arginine, asparagine, or cysteine residues in target proteins. This post-translational protein modification is the mechanism by which cholera-toxin and other bacterial toxins cause pathology in human host cells. Molecular cloning has identified five toxin-related GPI-anchored cell surface ARTs in the mouse (ART1, ART2.1, ART2.2, ART3, and ART4) and three in the human (ART1, ART3, and ART4). ART2—which has sparked interest because of its ability to activate the cytolytic P2X7 purinergic receptor by ADP-ribosylation—is encoded by two functional gene copies in the mouse genome while the human genome carries two inactivated ART2 pseudogenes. We generated stable transfectants for FLAG-tagged versions of each of the functional human and mouse ARTs. Using genetic immunization we raised monoclonal antibodies that recognize the native human ARTs on the surface of living cells. Some of these mAbs recognize an epitope shared with the mouse ART orthologue but not with more distant ART paralogues. Screening of primary cells and established cell lines by FACS revealed expression of ART1 by monocytes, neutrophils and myeloid leukemia cell lines but not by cell lines derived from solid tumors. ART1 and ART4 have been assigned the designations: CD296, and CD297, respectively.

Adenosine diphosphate (ADP)–ribosyl-transferases (ARTs) transfer ADP-ribose from nicotinamide adenine dinucleotide (NAD) onto target proteins. T cells express ART2.2, a toxin-related, glycosylphosphatidylinositol (GPI)–anchored ecto-enzyme. After the release of NAD from cells, ART2.2 ADP-ribosylates the P2X7 purinoceptor, lymphocyte function–associated antigen (LFA-1), and other membrane. Using lymphoma transfectants expressing either ART2.2 with its native GPI anchor (ART2.2-GPI) or ART2.2 with a grafted transmembrane anchor (ART2.2-Tm), we demonstrated that ART2.2-GPI but not ART2.2-Tm associated with glycosphingolipid-enriched microdomains (lipid rafts). At limiting substrate concentrations, ART2.2-GPI exhibited more than 10-fold higher activity than ART2.2-Tm. On intact cells, ART2.2-GPI ADP-ribosylated a small number of distinct target proteins. Strikingly, the disruption of lipid rafts by cyclodextrin or membrane solubilization by Triton X-100 increased the spectrum of modified target proteins. However, ART2.2 itself was a prominent target for ADP-ribosylation only when GPI anchored. Furthermore, cholesterol depletion or detergent solubilization abolished the auto-ADP-ribosylation of ART2.2. These findings imply that ART2.2-GPI, but not ART2.2-Tm, molecules are closely associated on the plasma membrane and lend support to the hypothesis that lipid rafts exist on living cells as platforms to which certain proteins are admitted and others are excluded. Our results further suggest that raft association focuses ART2.2 on specific targets that constitutively or inducibly associate with lipid rafts.

Mammalian ecto ADP-ribosyltransferases (ARTs) constitute a family of structurally related proteins expressed on the cell surface or secreted in the extracellular compartment. Using NAD+ as substrate, they transfer ADP-ribose groups onto target proteins. In contrast to intracellular poly(ADP-ribosyl)transferases (PARPs), these enzymes transfer a single ADPR and are thus mono-ARTs. Five paralogs (ART1-5) have been cloned but only four of them are expressed in human due to a defective ART2 gene, and six in the mouse as the result of ART2 gene duplication. The recent determination of the crystal structure of rat ART2 reveals homologies with bacterial ART toxins and provides a molecular basis for understanding the specificity of ARTs for their targets. A combination of different technological approaches reveals that ecto-ARTs are expressed in different tissues with privileged sites such as heart and skeletal muscles for ART1, T lymphocytes for ART2 or testis for ART5. It also indicates that ART expression is highly regulated. ADP-ribosylation of target proteins on cell surfaces or circulating in body fluids leads to reversible post-translational modifications which can inhibit the targets, as known for bacterial ARTs, or activate them, as in the crosstalk between mouse ART2 and the cytolytic P2X7 receptor on T lymphocytes. ART activity in the extracellular compartment provides sophisticated regulatory mechanisms for cell communication. This designates ecto-ARTs as new candidates for drug targeting.

The P2X7 receptor (P2X7R) is an ATP-gated channel that mediates apoptosis of cells of the immune system. The capacity of P2X7R to form large pores depends on its large cytoplasmic tail, which harbors a putative TNFR-related death domain. Previous transfection studies indicated that mouse P2X7R forms pores much less efficiently than its counterparts from humans and rats. In this study, we demonstrate that an allelic mutation (P451L) in the predicted death domain of P2X7R confers a drastically reduced sensitivity to ATP-induced pore formation in cells from some commonly used strains of mice, i.e., C57BL/6 and DBA/2. In contrast, most other strains of mice, including strains derived from wild mice, carry P451 at this position as do rats and humans. The effects of the P451L mutation resemble those of the E496A mutation in human P2X7R. These P2X7R mutants may provide useful tools to decipher the molecular mechanisms leading to pore formation.

Dlm-1 is a recently described gene which is upregulated in murine stromal cells lining tumors. The function of the 40 kDa DLM-1 protein is poorly understood. DLM-1 contains an N-terminal Z-DNA binding domain homologous to the Zalpha domain in the RNA editing enzyme ADAR1. We report the cloning of human and rat DLM-1. In addition to the Zalpha domain, three further conserved regions were identified. One of these is homologous to the second Z-DNA binding domain, Zbeta, of ADAR1. We find that human DLM-1 is predominantly expressed in lymphatic tissues. The gene spans 17 kb and consists of 10 exons. DNA transcripts are extremely heterogeneous as a result of alternative splicing and the usage of exon variants combined with at least two transcriptional start sites and 3'-terminal exons. The exon coding for the Zalpha domain was present in approximately one-third of the analyzed mRNAs. Nearly half of the transcripts contained exon variants that had premature stop codons incorporated. Based on our analysis, over 2000 different mRNAs may be produced due to alternative splicing and usage of different 5' and 3' ends. The cellular function of DLM-1 appears to call for a high degree of adaptation by this complex regulation.

The utility of nanobodies and conventional antibodies for in vivo imaging is well known, but optimum dosing and timing schedules for one versus the other have not been established. We aimed to improve specific tumor imaging in vivo with nanobodies and conventional antibodies using near-infrared fluorescence (NIRF) imaging. We used ARTC2 expressed on lymphoma cells as a model target antigen. ARTC2-specific nanobody s+16a and conventional antibody Nika102 were labeled with NIRF-dye AF680. In vivo NIRF-imaging of ARTC2-positive and ARTC2-negative xenografts was performed over 24 h post-injection of 5, 10, 25, or 50 µg of each conjugate. Specific target-binding and tissue-penetration were verified by NIRF imaging ex vivo, flow cytometry and fluorescence microscopy. NIRF-imaging of s+16a(680) in vivo revealed a six times faster tumor accumulation than of Nika102(680). Using 50 µg of s+16a(680) increased the specific signals of ARTC2-positive tumors without increasing background signals, allowing a tumor-to-background (T/B) ratio of 12.4 ± 4.2 within 6 h post-injection. Fifty micrograms of Nika102(680) increased specific signals of ARTC2-positive tumors but also of ARTC2-negative tumors and background, thereby limiting the T/B ratio to 6.1 ± 2.0. Ten micrograms of Nika102(680) only slightly reduced specific tumor signals but dramatically reduced background signals. Ex vivo analyses confirmed a faster and deeper tumor penetration with s+16a(680). Using nanobody s+16a allowed same-day imaging with a high T/B ratio, whereas antibody Nika102 gave optimal imaging results only 24 h post injection. Nanobody s+16a required a high dose, whereas antibody Nika102 had the best T/B-ratio at a low dose. Therefore, timing and dosage should be addressed when comparing nanobodies and conventional antibodies for molecular imaging purposes.

ADP-ribosyltransferases comprise a family of enzymes originally discovered as bacterial toxins and later characterised also in mammals. In mice, the ADP-ribosyltransferase ARTC2.2 is expressed at the surface of T lymphocytes and has been studied extensively. In the presence of extracellular NAD(+), ARTC2.2 ADP-ribosylates several cell surface target proteins and thereby regulates their function. P2X7, an ATP-gated cation channel, has been discovered as a prominent ARTC2.2 target at the surface of mouse T cells. ADP-ribosylation of P2X7 in the presence of low micromolar extracellular NAD(+) induces long-lasting P2X7 activation and triggers cell death. Regulatory T cell subsets (Tregs and NKT cells) are remarkably sensitive to NAD(+)-induced cell death (NICD). Thus, liberation of endogenous NAD(+) by stressed cells is now viewed as a danger signal promoting immune responses by hindering regulatory T cells. This review will highlight the recent discoveries on the in vivo role of the ARTC2.2/P2X7 pathway triggered by the endogenous release of extracellular NAD(+), the relative sensitivity of lymphocytes subsets to this regulatory pathway and its pharmacological manipulation using camelid-derived ARTC2.2-blocking nanobodies.

Lymphocytes express a number of NAD-metabolizing ectoenzymes, including mono(ADP-ribosyl)transferases (ART) and ADP ribosylcyclases. These enzymes may regulate lymphocyte functions following the release of NAD in injured or inflammatory tissues We report here that extracellular NAD induces apoptosis in BALB/c splenic T cells with an IC50 of 3–5 μM. Annexin V staining of cells was observed already 10 min after treatment with NAD in the absence of any additional signal. Removal of GPI-anchored cell surface proteins by phosphatidylinositol-specific phospholipase C treatment rendered cells resistant to NAD-mediated apoptosis. RT-PCR analyses revealed that resting BALB/c T cells expressed the genes for GPI-anchored ART2.1 and ART2.2 but not ART1. ART2-specific antisera blocked radiolabeling of cell surface proteins with both [32P]NAD and NAD-mediated apoptosis. Further analyses revealed that natural knockout mice for Art2.a (C57BL/6) or Art2.b (NZW) were resistant to NAD-mediated apoptosis. Labeling with [32P]NAD revealed strong cell surface ART activity on T cells of C57BL/6 and little if any activity on cells of NZW mice. T cells of (C57BL/6 × NZW)F1 animals showed strong cell surface ART activity and were very sensitive to NAD-induced apoptosis. As in BALB/c T cells, ART2-specific antisera blocked cell surface ART activity and apoptosis in (C57BL/6 × NZW)F1 T cells. The fact that T cells of F1 animals are sensitive to rapid NAD-induced apoptosis suggests that this effect requires the complementation of (at least) two genetic components. We propose that one of these is cell surface ART2.2 activity (defective in the NZW parent), the other a downstream effector of ADP-ribosylation (defective in the C57BL/6 parent).

The Salmonella enterica virulence-associated protein SpvB was recently shown to contain a carboxy-terminal mono(ADP-ribosyl)transferase domain. We demonstrate here that the catalytic domain of SpvB as well bacterial extracts containing full-length SpvB modifies a 43 kDa protein from macrophage-like J774-A.1 and epithelial MDCK cells as shown by label transfer from [32P]-nicotinamide adenine dinucleotide (NAD) to the 43 kDa protein. When analysed by two-dimensional gel electrophoresis, the same protein was modified in cells infected with S. enterica serovariant Dublin strain SH9325, whereas infection with an isogenic spvB mutant strain did not result in modification. Immunoprecipitation and immunoblotting experiments using SH9325-infected cells identified the modified protein as actin. The isolated catalytic domain of SpvB mediated transfer of 32P from [32P]-NAD to actins from various sources in vitro, whereas isolated eukaryotic control proteins or bacterial proteins were not modified. In an in vitro actin polymerization assay, the isolated catalytic SpvB domain prevented the conversion of G actin into F actin. Microscopic examination of MDCK cells infected with SH9325 revealed morphological changes and loss of filamentous actin content, whereas cells infected with the spvB mutant remained virtually unaffected. We conclude that actin is a target for an SpvB-mediated modification, most probably ADP-ribosylation, and that the modification of G actin interferes with actin polymerization.

Release of NAD+ during preparation of murine lymphocytes causes enzymatic ADP-ribosylation of cell-surface proteins on T cells, catalyzed by toxin-related ecto-ADP-ribosyltransferase, ARTC2. ADP-riboslyation activates the cytolytic P2X7 ion channel and affects, in particular, the vitality and function of Tregs and NKT cells. Here, we describe a simple method—injection of an ARTC2-blocking nanobody—to greatly improve Treg and NKT cell vitality and to preserve their function during in vitro assays and in adoptive-transfer experiments. Moreover, we present a method for the sorting of functional, primary NKT cells, based on coexpression of ARTC2 and NK1.1. Our results pave the way for the efficient ex vivo proliferation of Tregs and NKT cells and for new experimental and therapeutic uses of these important regulatory cells.

CD38, adenosine-5′-diphosphate-ribosyl cyclase 1, is a multifunctional enzyme, expressed on a wide variety of cell types. CD38 has been assigned diverse functions, including generation of calcium-mobilizing metabolites, cell activation, and chemotaxis. Using a murine Listeria monocytogenes infection model, we found that CD38 knockout (KO) mice were highly susceptible to infection. Enhanced susceptibility was already evident within 3 days of infection, suggesting a function of CD38 in the innate immune response. CD38 was expressed on neutrophils and inflammatory monocytes, and especially inflammatory monocytes further upregulated CD38 during infection. Absence of CD38 caused alterations of the migration pattern of both cell types to sites of infection. We observed impaired accumulation of cells in the spleen but surprisingly similar or even higher accumulation of cells in the liver. CD38 KO and wild-type mice showed similar changes in the composition of neutrophils and inflammatory monocytes in blood and bone marrow, indicating that mobilization of these cells from the bone marrow was CD38 independent. In vitro , macrophages of CD38 KO mice were less efficient in uptake of listeria but still able to kill the bacteria. Dendritic cells also displayed enhanced CD38 expression following infection. However, absence of CD38 did not impair the capacity of mice to prime CD8 + T cells against L. monocytogenes , and CD38 KO mice could efficiently control secondary listeria infection. In conclusion, our results demonstrate an essential role for CD38 in the innate immune response against L. monocytogenes .

Mono-ADP-ribosylation was originally discovered as the mechanism by which diphtheria toxin inactivates protein synthesis. It is now apparent that higher organisms use analogous mechanisms to regulate endogenous metabolism. Here, the relationship between bacterial toxins and vertebrate mono-ADP ribosyltransferases is explored.

The CD8αβ coreceptor is crucial for effective peptide: MHC-I recognition by the TCR of CD8(+) T cells. Adenosine diphosphate ribosyl transferase 2.2 (ART2.2) utilizes extracellular NAD(+) to transfer ADP-ribose to arginine residues of extracellular domains of surface proteins. Here, we show that in the presence of extracellular NAD(+) , ART2.2 caused ADP-ribosylation of CD8-β on murine CD8(+) T cells in vitro and in vivo. Treatment with NAD(+) prevented binding of anti-CD8-β mAb YTS156.7.7 but not of mAb H35-17.2, indicating that NAD(+) caused modification of certain epitopes and not a general loss of CD8-β. Loss of antibody binding was strictly dependent on ART2.2, because it was not observed on ART2-deficient T cells or in the presence of inhibitory anti-ART2.2 single-domain antibodies. ADP-ribosylation of CD8-β occurred during cell isolation, particularly when cells were isolated from CD38-deficient mice. Incubation of ART2-expressing, but not of ART2-deficient, OVA-specific CD8(+) T cells with NAD(+) interfered with binding of OVA257-264 :MHC-I tetramers. In line with this result, treatment of WT mice with NAD(+) resulted in reduced CD8(+) T-cell mediated cytotoxicity in vivo. We propose that ADP-ribosylation of CD8-β can regulate the coreceptor function of CD8 in the presence of elevated levels of extracellular NAD(+) .

P2X7 is a homotrimeric ion channel with two transmembrane domains and a large extracellular ATP-binding domain. It plays a key role in the response of immune cells to danger signals released from cells at sites of inflammation. Gating of murine P2X7 can be induced by the soluble ligand ATP, as well as by NAD(+)-dependent ADP-ribosylation of arginine 125, a posttranslational protein modification catalyzed by the toxin-related ecto-enzymes ART2.1 and ART2.2. R125 is located at the edge of the ligand-binding crevice. Recently, an alternative splice variant of P2X7, designated P2X7(k), was discovered that differs from the previously described variant P2X7(a) in the N-terminal 42 amino acid residues composing the first cytosolic domain and most of the Tm1 domain. Here we compare the two splice variants of murine P2X7 with respect to their sensitivities to gating by ADP-ribosylation in transfected HEK cells. Our results show that the P2X7(k) variant is sensitive to activation by ADP-ribosylation whereas the P2X7(a) variant is insensitive, despite higher cell surface expression levels. Interestingly, a single point mutation (R276K) renders the P2X7(a) variant sensitive to activation by ADP-ribosylation. Residue 276 is located at the interface of neighboring subunits approximately halfway between the ADP-ribosylation site and the transmembrane domains. Moreover, we show that naive and regulatory T cells preferentially express the more sensitive P2X7(k) variant, while macrophages preferentially express the P2X7(a) variant. Our results indicate that differential splicing of alternative exons encoding the N-terminal cytosolic and transmembrane domains of P2X7 control the sensitivity of different immune cells to extracellular NAD(+) and ATP.

RT6 is a glycosyl phosphatidylinositol-anchored cell membrane protein, whose expression is restricted to peripheral T cells and intraepithelial lymphocytes. It has attracted interest as a T cell differentiation marker and activation antigen in rats. The only known protein to which RT6 shows significant homology is a recently cloned mono(ADP-ribosyl)transferase of rabbit skeletal muscle which is distantly related also to certain bacterial toxins. Intriguingly, whereas the rat carries a single copy RT6 gene with two known highly divergent alleles, the mouse carries two closely linked, functional RT6 genes that show approximately 85% sequence identity. We have now cloned and sequenced the homologues of the RT6 genes from humans of distinct ethnic backgrounds and of the chimpanzee. Surprisingly, in each case, three premature in-frame stop codons preclude expression of the single copy RT6 gene as a cell surface protein. Otherwise, the RT6 genes of human and chimpanzee exhibit high structural conservation to their rodent counterparts. RNA expression analyses indicate that the RT6 gene is not transcriptionally active in human T cells or any other human tissue analyzed so far. To our knowledge, RT6 represents the first mammalian membrane protein identified that has been lost universally in the human and chimpanzee species due to gene inactivation.

The objective of this study was to quantify enzymatic activity on the surface of T cells by magnetic resonance imaging (MRI) using R2 and R2* relaxometry. Lymphoma cells expressing adenosine diphosphate (ADP)-ribosyltransferase 2 (ART2) were incubated with increasing doses of its substrate etheno–nicotinamide adenine dinucleotide (NAD), resulting in increasing amounts of surface protein ADP-ribosylation. Etheno-ADP-ribosylated proteins were detected with monoclonal antibody 1G4 and superparamagnetic iron oxide conjugated secondary antibodies (Ab-SPIO). Labeling efficiency was determined with R2 and R2* relaxometry on a clinical 3.0 T scanner. Parallel aliquots of cells were analyzed by flow cytometry. Cell-bound SPIO conjugates were detected by immunofluorescence and electron microscopy and quantified by atomic absorption spectroscopy. To mimic an inflammatory site in vivo, Ab-SPIO-labeled cells were injected subcutaneously in mice and analyzed by MRI. Immunofluorescence and electron microscopy confirmed cell-surface localization of Ab-SPIO. MRI of Ab-SPIO-labeled cells showed a corresponding signal reduction. Increases in R2 and R2* determined by magnetic resonance relaxometry correlated linearly with the expression level of ART2 and the concentration of the ART2 substrate etheno-NAD. R2 and R2* increases correlated linearly with the results from flow cytometry and atomic absorption spectroscopy analyses. Quantitative R2 and R2* mapping enable noninvasive determination of enzymatic activity on T cells and holds promise for characterization of inflammatory sites in vivo by MRI.

Extracellular NAD(+) and ATP trigger the shedding of CD62L and the externalization of phosphatidylserine on murine T cells. These events depend on the P2X(7) ion channel. Although ATP acts as a soluble ligand to activate P2X(7), gating of P2X(7) by NAD(+) requires ecto-ADP-ribosyltransferase ART2.2-catalyzed transfer of the ADP-ribose moiety from NAD(+) onto Arg125 of P2X(7). Steady-state concentrations of NAD(+) and ATP in extracellular compartments are highly regulated and usually are well below the threshold required for activating P2X(7). The goal of this study was to identify possible endogenous sources of these nucleotides. We show that lysis of erythrocytes releases sufficient levels of NAD(+) and ATP to induce activation of P2X(7). Dilution of erythrocyte lysates or incubation of lysates at 37 degrees C revealed that signaling by ATP fades more rapidly than that by NAD(+). We further show that the routine preparation of primary lymph node and spleen cells induces the release of NAD(+) in sufficient concentrations for ART2.2 to ADP-ribosylate P2X(7), even at 4 degrees C. Gating of P2X(7) occurs when T cells are returned to 37 degrees C, rapidly inducing CD62L-shedding and PS-externalization by a substantial fraction of the cells. The "spontaneous" activation of P2X(7) during preparation of primary T cells could be prevented by i.v. injection of either the surrogate ART substrate etheno-NAD or ART2.2-inhibitory single domain Abs 10 min before sacrificing mice.

Extracellular ATP mediates a diverse array of biological responses in many cell types and tissues, including immune cells. We have demonstrated that ATP induces purinergic receptor P2X(7) mediated membrane permeabilization, apoptosis, and cytokine expression in murine mast cells (MCs). Here, we report that MCs deficient in the expression of the P2X(7) receptor are resistant to the ATP-induced membrane permeabilization and apoptosis. However, ATP affects the tyrosine phosphorylation pattern of P2X(7)knockout cells, leading to the activation of ERK1/2. Furthermore, ATP induces expression of several cytokines and chemokines in these cells, including IL-4, IL-6, IFN-gamma, TNF-alpha, RANTES, and MIP-2, at the mRNA level. In addition, the release of IL-6 and IL-13 to cell-conditioned medium was confirmed by ELISA. The ligand selectivity and pharmacological profile indicate the involvement of two P2X family receptors, P2X(1) and P2X(3). Thus, depending on genetic background, particular tissue microenvironment, and ATP concentration, MCs can presumably engage different P2X receptor subtypes, which may result in functionally distinct biological responses to extracellular nucleotides. This finding highlights a novel level of complexity in the sophisticated biology of MCs and may facilitate the development of new therapeutic approaches to modulate MC activities.

Mono(ADP-ribosyl)transferases regulate the function of target proteins by attaching ADP-ribose to specific amino acid residues in the proteins. We have characterized the gene for mouse arginine-specific ADP-ribosyltransferase, Art1. Southern blot analyses indicate that Art1 is a single-copy gene. Northern blot and reverse transcription–PCR analyses demonstrate prominent expression of Art1 in cardiac and skeletal muscle, and lower levels in spleen, lung, liver and fetal tissues. While human ART1 is not represented in the public expressed sequence tag (EST) database, the database contains 14 mouse Art1 ESTs. The Art1 gene encompasses four exons spanning 20 kb of genomic DNA. The deduced amino acid sequence of Art1 exhibits the characteristic features of a glycosylphosphatidylinositol-anchored membrane protein. It shows 75–77% sequence identity with its orthologues from the human and rabbit, and 33–34% identity with its paralogues from the mouse, Art2-1 and Art2-2. Separate exons encode the N- and C-terminal signal peptides, and a single long exon encodes the entire predicted native polypeptide chain. We expressed Art1 in 293T cells as a recombinant fusion protein with the Fc portion of human IgG1. This soluble protein exhibits enzyme activities characteristic of arginine-specific ADP-ribosyltransferases. The availability of the Art1 gene provides the basis for applying transgene and knockout technologies to further probe the function of this gene product.

The chemokine receptor CXCR3 is highly expressed on Th1 polarized T cells and has been predicted to play an important role in T cell recruitment and immune response in a number of inflammatory and autoimmune diseases. For testing whether CXCR3 plays a role in renal inflammation, CXCR3-deficient mice were generated and nephrotoxic nephritis was induced in C57BL/6 CXCR3(-/-) and C57BL/6 wild-type mice. Induction of the nephrotoxic nephritis leads to an increased renal mRNA expression of IP-10/CXCL10 (8.6-fold), Mig/CXCL9 (2.3-fold), and I-TAC/CXCL11 (4.9-fold) during the autologous phase at days 7 and 14. This increased chemokine expression was paralleled by the renal infiltration of T cells, followed by renal tissue injury, albuminuria, and loss of renal function. Compared with wild-type mice, CXCR3-deficient mice had significantly reduced renal T cell infiltrates. Moreover, CXCR3(-/-) mice developed less severe nephritis, with significantly lower albuminuria, better renal function, and a reduced frequency of glomerular crescent formation. Nephritic wild-type and CXCR3(-/-) mice both elicited an efficient systemic nephritogenic immune response in terms of antigen-specific IgG production and IFN-gamma expression by splenocytes in response to the nephritogenic antigen. These findings indicate that the ameliorated nephritis in CXCR3-deficient mice is due to impaired renal trafficking of effector T cells rather than their inability to mount an efficient humoral or cellular immune response. The neutralization of CXCR3 might be a promising therapeutic strategy for Th1-dependent inflammatory renal disease.

Many bacterial toxins kill animal cells by adenosine diphosphate (ADP)-ribosylating intracellular target proteins. Mammalian cells express toxin-related cell surface ADP-ribosyltransferases (ARTs) that transfer ADP-ribose from nicotinamide adenine dinucleotide (NAD) onto arginine residues of other membrane proteins. The association of these glycosylphosphatidylinositol (GPI)-anchored ectoenzymes with glycolipid rafts focuses them onto components of the signal transduction machinery. Exposing murine T cells to NAD, the ART substrate, induces a cascade of reactions that culminates in cell death by apoptosis. This mechanism, dubbed 'NAD-induced cell death' or NICD, is initiated when ART2 ADP-ribosylates the cytolytic P2X7 purinergic receptor, inducing formation of a cation channel, opening of a nonselective pore, shedding of CD62L from the cell surface, exposure of phosphatidylserine on the outer leaflet of the plasma membrane, breakdown of the mitochondrial membrane potential, and DNA-fragmentation. The ART substrate NAD is produced in large amounts inside the cell and can be released from damaged cells during inflammation and tissue injury. In the extracellular environment, the signaling function of NAD is terminated by NAD-degrading ectoenzymes such as CD38. We propose that ART2-catalyzed ADP-ribosylation of P2X7 represents the paradigm of a regulatory mechanism by which ART-expressing cells can sense and respond to the release of NAD from damaged cells.

Background ADP-ribosylation is an enzyme-catalyzed posttranslational protein modification in which mono(ADP-ribosyl)transferases (mARTs) and poly(ADP-ribosyl)transferases (pARTs) transfer the ADP-ribose moiety from NAD onto specific amino acid side chains and/or ADP-ribose units on target proteins. Results Using a combination of database search tools we identified the genes encoding recognizable pART domains in the public genome databases. In humans, the pART family encompasses 17 members. For 16 of these genes, an orthologue exists also in the mouse, rat, and pufferfish. Based on the degree of amino acid sequence similarity in the catalytic domain, conserved intron positions, and fused protein domains, pARTs can be divided into five major subgroups. All six members of groups 1 and 2 contain the H-Y-E trias of amino acid residues found also in the active sites of Diphtheria toxin and Pseudomonas exotoxin A, while the eleven members of groups 3 – 5 carry variations of this motif. The pART catalytic domain is found associated in Lego-like fashion with a variety of domains, including nucleic acid-binding, protein-protein interaction, and ubiquitylation domains. Some of these domain associations appear to be very ancient since they are observed also in insects, fungi, amoebae, and plants. The recently completed genome of the pufferfish T. nigroviridis contains recognizable orthologues for all pARTs except for pART7. The nearly completed albeit still fragmentary chicken genome contains recognizable orthologues for twelve pARTs. Simpler eucaryotes generally contain fewer pARTs: two in the fly D. melanogaster, three each in the mosquito A. gambiae, the nematode C. elegans, and the ascomycete microfungus G. zeae, six in the amoeba E. histolytica, nine in the slime mold D. discoideum, and ten in the cress plant A. thaliana. GenBank contains two pART homologues from the large double stranded DNA viruses Chilo iridescent virus and Bacteriophage Aeh1 and only a single entry (from V. cholerae) showing recognizable homology to the pART-like catalytic domains of Diphtheria toxin and Pseudomonas exotoxin A. Conclusion The pART family, which encompasses 17 members in the human and 16 members in the mouse, can be divided into five subgroups on the basis of sequence similarity, phylogeny, conserved intron positions, and patterns of genetically fused protein domains.

ADP-ribosyltransferase-2 (ART2), a GPI-anchored, toxin-related ADP-ribosylating ectoenzyme, is prominently expressed by murine T cells but not by B cells. Upon exposure of T cells to NAD, the substrate for ADP-ribosylation, ART2 catalyzes ADP-ribosylation of the P2X7 purinoceptor and other functionally important cell surface proteins. This in turn activates P2X7 and induces exposure of phosphatidylserine and shedding of CD62L. CD38, a potent ecto-NAD-glycohydrolase, is strongly expressed by most B cells but only weakly by T cells. Following incubation with NAD, CD38-deficient splenocytes exhibited lower NAD-glycohydrolase activity and stronger ADP-ribosylation of cell surface proteins than their wild-type counterparts. Depletion of CD38high cells from wild-type splenocytes resulted in stronger ADP-ribosylation on the remaining cells. Similarly, treatment of total splenocytes with the CD38 inhibitor nicotinamide 2′-deoxy-2′-fluoroarabinoside adenine dinucleotide increased the level of cell surface ADP-ribosylation. Furthermore, the majority of T cells isolated from CD38-deficient mice “spontaneously” exposed phosphatidylserine and lacked CD62L, most likely reflecting previous encounter with ecto-NAD. Our findings support the notion that ecto-NAD functions as a signaling molecule following its release from cells by lytic or nonlytic mechanisms. ART2 can sense and translate the local concentration of ecto-NAD into corresponding levels of ADP-ribosylated cell surface proteins, whereas CD38 controls the level of cell surface protein ADP-ribosylation by limiting the substrate availability for ART2.

ART4 (CD297) is a member of the family of toxin-related ADP-ribosyltransferases (ARTs) and is the carrier of the Dombrock blood group alloantigens (Do). Two mouse monoclonal antibodies (MIMA-52 and MIMA-53), and two rat monoclonal antibodies (N0NI-B4 and NONI-B63) were obtained following immunization of mice with human Do/ART4-transfected cells and of rats with human Do/ART4 cDNA, respectively. All four mAbs recognize Do/ART4-transfected Jurkat cells but not untransfected cells by FACS analysis. Staining of Do/ART4-transfected cells by these mAbs was reduced following treatment of cells with PI-PLC, confirming that Do/ART4 is anchored in the cell membrane by linkage to glycosylphosphatidylinositol as predicted from its amino acid sequence. The four mAbs did not react with Gy(a-) (Dombrock null) erythrocytes but agglutinated other red blood cells. By flow cytometric analysis, all mAbs reacted prominently with erythrocytes, and weakly with peripheral blood monocytes and splenic macrophages, but not with B-lymphocytes or T-lymphocytes. The mAbs reacted weakly also with human umbilical vein endothelial cells and the basophilic leukemia KU-812. Immunohistology revealed staining of epithelia and endothelia on sections of tonsils. In FACS analyses NONI-B4 competed with MIMA-52 for binding to Do/ART4-transfected cells and erythrocytes, whereas NONI-B63 competed with MIMA-53. Neither of the mAbs reacted with mouse ART4-transfected cells, but NONI-B63 and MIMA-53 did react with a mouse/human ART4 chimera, indicating that the epitope recognized by these mAbs lies in the C-terminal half of the protein.

T cells express a toxin-related ADP-ribosylating ectoenzyme, ART2. Exposure of mature T cells to NAD, the substrate for ADP-ribosylation, induces cell death. ART2-catalyzed ADP-ribosylation activates the cytolytic P2X7 purinoceptor, causing calcium flux, pore formation, phosphatidylserine exposure, shedding of CD62L, cell shrinkage, and propidium iodide uptake. Interestingly, much lower NAD than ATP concentrations are required to activate P2X7. NAD-induced cell death (NICD) operates with endogenous sources of NAD released upon cell lysis. These findings identify P2X7 as a key effector of NICD and demonstrate that P2X7 can be activated by an endogenous ligand other than ATP. Our results delineate an alternative mechanism for inducing T cell death and set an interesting precedent for immunoregulation via crosstalk between NAD-dependent ADP-ribosyltransferases and purinoceptors.

NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.