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Introduction: The serine/threonine kinase 17B (STK17B) is involved in setting the threshold for T cell activation and its absence sensitizes T cells to suboptimal stimuli. Consequently, STK17B represents an attractive potential target for cancer immunotherapy., Methods: To assess the potential of STK17B as an immuno-oncology target, we developed potent and selective tool compounds from starting points in Blueprint Medicines Corporation's proprietary kinase inhibitor library. To characterize these molecules, enzyme and cellular assays for STK17A and STK17B were established to drive chemistry optimization. Mass spectrometry-based phosphoproteomics profiling with tool inhibitors led to the identification of Ser19 on myosin light chain 2 as STK17B substrate, which is then developed into a flow cytometry-based pharmacodynamic readout of STK17B inhibition both in vitro and in vivo ., Results: In a mouse T cell activation assay, STK17B inhibitors demonstrated the ability to enhance interleukin-2 (IL-2) production. Similarly, treatment with STK17B inhibitors resulted in stronger cytokine secretion in human T cells activated using a T cell bispecific antibody. Subsequent chemistry optimization led to the identification of a highly selective and orally bioavailable tool compound, BLU7482. In vivo , STK17B inhibition led to dose-dependent modulation of myosin light chain 2 phosphorylation and enhanced priming of naïve T cells, as determined by upregulation of CD69, IL-2 and interferon-γ secretion. In line with increased T cell activation, treatment with STK17B inhibitor enhanced antitumor activity of anti-PD-L1 antibody in the MCA205 model., Conclusions: In summary, we successfully identified and optimized STK17B kinase inhibitors which led to increased T cell responses in vitro and in vivo . This allowed us to evaluate the potential of STK17B inhibition as an approach for cancer immunotherapy., Competing Interests: Authors FS, JEC, RC, CDS, JG, JLK, CK, KM, RM, EP, YT, TT, RW, RW, and MD were employed by the company Blueprint Medicines Corporation at the time of the study. FS, JG, RC, JK, CK, KM, EP, YT, RM, TT, and RW hold stocks and shares in Blueprint Medicines Corporation. Authors FR, JE, HF, LG, PJ, AM, UH, ST, MT, and JRB were employed by the company F. Hoffmann-La Roche Ltd. at the time of the study. FR and JRB hold stocks in F. Hoffmann-La Roche Ltd. JEC is currently affiliated with Reverie Labs, Cambridge, MA, USA. CDS is currently affiliated with Curie.Bio, Boston, MA, USA. NP is currently affiliated with ROME therapeutics, Boston, MA, USA. ST is currently affiliated with Altos Labs, Los Altos, CA, USA. RW is currently an independent consultant. MD is currently affiliated with Atavistik Bio, Cambridge, MA, USA. The authors declare that this research received funding from Blueprint Medicines Corporation and F. Hoffmann-La Roche Ltd. The sponsor was involved in the study design, collection, analysis, and interpretation of data, as well as data checking of information provided in the manuscript., (Copyright © 2024 Scheuplein, Renner, Campbell, Campbell, De Savi, Eckmann, Fischer, Ge, Green, Jakob, Kim, Kinkema, McGinn, Medina, Müller, Perez, Perola, Timsit, Traore, Hopfer, Tyanova, Tzouros, Wang, Woessner, Dorsch and Bischoff.)

Invariant natural killer T cells (iNKTs) directly kill tumor cells and trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK) cells, and T and B cells. As such, iNKTs serve as a powerful tool for use in cell-based cancer immunotherapy. iNKT cell activation commonly requires engagement of the invariant T cell receptor (iTCR) by CD1d presenting glycolipid antigens. However, transformed cells often down-regulate CD1d expression, which results in a reduction of iNKT cell anti-tumor functions. One approach to circumvent this critical barrier to iNKT cell activation is to develop an agonistic antibody that binds directly to the iTCR without the requirement for CD1d-mediated antigen presentation. To this end, we have characterized the iNKT cell stimulatory properties of NKTT320, a novel, recombinant, humanized, monoclonal antibody that binds selectively and with high affinity to human iTCRs. Strikingly, immobilized NKTT320 mediated robust iNKT cell activation (upregulation of CD25 and CD69) and proliferation (carboxyfluorescein succinimidyl ester (CFSE) dilution), as well as Th1 and Th2 cytokine production. Additionally, iNKTs stimulated by plate-bound NKTT320 exhibited increased intracellular levels of granzyme B and degranulation (exposure of CD107 on the cell surface). Furthermore, both soluble and immobilized NKTT320 induced iNKT cell-mediated activation of bystander immune cells, suggesting that this novel anti-iTCR antibody facilitates both direct and indirect iNKT cell cytotoxicity. These studies are significant, as they provide a framework by which iNKT cell anti-cancer functions could be enhanced for therapeutic purposes.

iNKT cells and mast cells have both been implicated in the syndrome of allergic asthma through their activation-induced release of Th2 type cytokines and secretion of histamine and other mediators, respectively, which can promote airways hyperresponsiveness (AHR) to agents such as methacholine. However, a mechanistic link between iNKT cells and mast cell recruitment or activation has never been explored. Our objective was to determine whether iNKT cells are necessary for the recruitment of mast cells and if iNKT cells can influence the acute allergen induced bronchoconstriction (AIB) caused by mast cell mediator release. To do so, we pharmacologically eliminated iNKT cells using a specific antibody (NKT-14) and examined its impact on airway inflammation and physiological phenotype. In mice treated with NKT-14, the elimination of iNKT cells was sufficient to prevent AHR and pulmonary eosinophilic inflammation elicited by administration of the iNKT cell agonist αGalCer. In mice treated with NKT-14 and then sensitized and challenged with house dust mite extract (HDM), eliminating the iNKT cells significantly reduced both AHR and AIB but did not affect pulmonary inflammation, the mast cell population, nor the release of the mast cell mediators mast cell protease-1 and prostaglandin D2. We conclude that while iNKT cells contribute to the phenotype of allergic airways disease through the manifestation of AIB and AHR, their presence is not required for mast cell recruitment and activation, or to generate the characteristic inflammatory response subsequent to allergen challenge., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Invariant Natural Killer T (iNKT) cells are a T cell subset expressing an invariant T Cell Receptor (TCR) that recognizes glycolipid antigens rather than peptides. The cells have both innate-like rapid cytokine release, and adaptive-like thymic positive selection. iNKT cell activation has been implicated in the pathogenesis of allergic asthma and inflammatory diseases, while reduced iNKT cell activation promotes infectious disease, cancer and certain autoimmune diseases such as Type 1 diabetes (T1D). Therapeutic means to reduce or deplete iNKT cells could treat inflammatory diseases, while approaches to promote their activation may have potential in certain infectious diseases, cancer or autoimmunity. Thus, we developed invariant TCR-specific monoclonal antibodies to better understand the role of iNKT cells in disease. We report here the first monoclonal antibodies specific for the mouse invariant TCR that by modifying the Fc construct can specifically deplete or activate iNKT cells in vivo in otherwise fully immuno-competent animals. We have used both the depleting and activating version of the antibody in the NOD model of T1D. As demonstrated previously using genetically iNKT cell deficient NOD mice, and in studies of glycolipid antigen activated iNKT cells in standard NOD mice, we found that antibody mediated depletion or activation of iNKT cells respectively accelerated and retarded T1D onset. In BALB/c mice, ovalbumin (OVA) mediated airway hyper-reactivity (AHR) was abrogated with iNKT cell depletion prior to OVA sensitization, confirming studies in knockout mice. Depletion of iNKT cells after sensitization had no effect on AHR in the conducting airways but did reduce AHR in the lung periphery. This result raises caution in the interpretation of studies that use animals that are genetically iNKT cell deficient from birth. These activating and depleting antibodies provide a novel tool to assess the therapeutic potential of iNKT cell manipulation.

Invariant Natural Killer T (iNKT) cells are a subset of T cells recognizing glycolipid antigens presented by CD1d. Human iNKT cells express a conserved T cell receptor (TCR)-α chain (Vα24-Jα18) paired with a specific beta chain, Vβ11. The cells are both innate-like, with rapid cytokine release, and adaptive-like, including thymic positive selection. Over activation of iNKT cells can mediate tissue injury and inflammation in multiple organ systems and play a role in mediating the pathology associated with clinically important inflammatory diseases. At the same time, iNKT cell activation can play a role in protecting against infectious disease and cancer or modulate certain autoimmune diseases through its impact on both the innate and adaptive immune system. This suggests that approaches to cause iNKT cell reduction and/or depletion could treat inflammatory diseases while approaches to promote activation may have therapeutic potential in certain infections, cancer or autoimmune disease. This report summarizes the characterization of a humanized monoclonal depleting antibody (NKTT120) in the cynomolgus macaque. NKTT120 is being developed to treat iNKT mediated inflammation that is associated with chronic inflammatory conditions like sickle cell disease and asthma. NKTT120 binds to human iTCRs and to FCγRI and FCγRIII and has been shown to kill target cells in an ADCC assay at low concentrations consistent with the FCγR binding. iNKT cells were depleted within 24 hours in cynomolgus macaques, but T cell, B cell, and NK cell frequencies were unchanged. iNKT cell recovery was dose and time dependent. T cell dependent antigen responses were not impaired by NKTT120 mediated iNKT depletion as measured by response to KLH challenge. NKTT120 administration did not induce an inflammatory cytokine release at doses up to 10 mg/kg. These data support the use of NKTT120 as an intervention in inflammatory diseases where iNKT reduction or depletion could be beneficial.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Although P2rx7 has been proposed as a type 1 diabetes (T1D) susceptibility gene in NOD mice, its potential pathogenic role has not been directly determined. To test this possibility, we generated a new NOD stock deficient in P2X(7) receptors. T1D development was not altered by P2X(7) ablation. Previous studies found CD38 knockout (KO) NOD mice developed accelerated T1D partly because of a loss of CD4(+) invariant NKT (iNKT) cells and Foxp3(+) regulatory T cells (Tregs). These immunoregulatory T cell populations are highly sensitive to NAD-induced cell death activated by ADP ribosyltransferase-2 (ART2)-mediated ADP ribosylation of P2X(7) receptors. Therefore, we asked whether T1D acceleration was suppressed in a double-KO NOD stock lacking both P2X(7) and CD38 by rescuing CD4(+) iNKT cells and Tregs from NAD-induced cell death. We demonstrated that P2X(7) was required for T1D acceleration induced by CD38 deficiency. The CD38 KO-induced defects in homeostasis of CD4(+) iNKT cells and Tregs were corrected by coablation of P2X(7). T1D acceleration in CD38-deficient NOD mice also requires ART2 expression. If increased ADP ribosylation of P2X(7) in CD38-deficient NOD mice underlies disease acceleration, then a comparable T1D incidence should be induced by coablation of both CD38 and ART2, or CD38 and P2X(7). However, a previously established NOD stock deficient in both CD38 and ART2 expression is T1D resistant. This study demonstrated the presence of a T1D resistance gene closely linked to the ablated Cd38 allele in the previously reported NOD stock also lacking ART2, but not in the newly generated CD38/P2X(7) double-KO line.

The ectoenzyme ADP-ribosyltransferase 2.2 (ART2.2) can apoptotically delete various T-cell subsets. Depending on the involved apoptotic T-cell subset, enhanced ART2.2 activity could result in immunosuppression or autoimmunity. Diminished activity of the CD38 ectoenzyme that normally represents a counter-regulatory competitor for the NAD substrate represents one mechanism enhancing ART2.2 activity. Hence, it would be desirable to develop an agent that efficiently blocks ART2.2 activity in vivo. While the llama derived recombinant s+16 single domain antibody overcame the difficulty of specifically targeting the ART2.2 catalytic site potential therapeutic use of this reagent is limited due to short in vivo persistence. Thus, we tested if a modified version of s+16 incorporating the murine IgG1 Fc tail (s+16Fc) mediated long-term efficient in vivo suppression of ART2.2. We reasoned an ideal model to test the s+16Fc reagent were NOD mice in which genetic ablation of CD38 results in an ART2.2 mediated reduction in already sub-normal numbers of immunoregulatory natural killer T-(NKT) cells to a level that no longer allows them when activated by the super-agonist alpha-galactosylceramide (alpha-GalCer) to elicit effects inhibiting autoimmune type 1 diabetes (T1D) development. Treatment with s+16Fc efficiently mediated long-term in vivo inhibition of ART2.2 activity in NOD.CD38(null) mice, restoring their iNKT cell numbers to levels that upon alpha-GalCer activation were capable of inhibiting T1D development., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Objective: In part, activation of invariant natural killer T (iNKT)-cells with the superagonist alpha-galactosylceramide (alpha-GalCer) inhibits the development of T-cell-mediated autoimmune type 1 diabetes in NOD mice by inducing the downstream differentiation of antigen-presenting dendritic cells (DCs) to an immunotolerogenic state. However, in other systems iNKT-cell activation has an adjuvant-like effect that enhances rather than suppresses various immunological responses. Thus, we tested whether in some circumstances genetic variation would enable activated iNKT-cells to support rather than inhibit type 1 diabetes development., Research Design and Methods: We tested whether iNKT-conditioned DCs in NOD mice and a major histocompatibility complex-matched C57BL/6 (B6) background congenic stock differed in capacity to inhibit type 1 diabetes induced by the adoptive transfer of pathogenic AI4 CD8 T-cells., Results: Unlike those of NOD origin, iNKT-conditioned DCs in the B6 background stock matured to a state that actually supported rather than inhibited AI4 T-cell-induced type 1 diabetes. The induction of a differing activity pattern of T-cell costimulatory molecules varying in capacity to override programmed death-ligand-1 inhibitory effects contributes to the respective ability of iNKT-conditioned DCs in NOD and B6 background mice to inhibit or support type 1 diabetes development. Genetic differences inherent to both iNKT-cells and DCs contribute to their varying interactions in NOD and B6.H2(g7) mice., Conclusions: This great variability in the interactions between iNKT-cells and DCs in two inbred mouse strains should raise a cautionary note about considering manipulation of this axis as a potential type 1 diabetes prevention therapy in genetically heterogeneous humans.

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.

The cytolytic P2X7 purinoceptor is widely expressed on leukocytes and has sparked interest because of its key role in the activation of the inflammasome, the release of the pro-inflammatory cytokine IL-1beta and cell death. We report here the functional characterisation of a R276A gain-of-function mutant analysed for its capacities to induce membrane depolarisation, calcium influx and opening of a large membrane pore permeable to YO-PRO-1. Our results highlight the particular sensitivity of R276A mutant to low micromolar adenosine triphosphate (ATP) concentrations, which possibly reflect an increased affinity for its ligands, and a slower closing kinetics of the receptor channel. Our findings support the notion that evolutionary pressures maintain the low sensitivity of P2X7 to ATP. We also believe that the R276A mutant described here may be useful for the generation of new animal models with exacerbated P2X7 functions that will serve to better characterise its role in inflammation and in immune responses.

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.

Objective: Although the H2(g7) major histocompatibility complex (MHC) provides the primary pathogenic component, the development of T-cell-mediated autoimmune type 1 diabetes in NOD mice also requires contributions from other susceptibility (Idd) genes. Despite sharing the H2(g7) MHC, the closely NOD-related NOR strain remains type 1 diabetes resistant because of contributions of protective Idd5.2, Idd9/11, and Idd13 region alleles. To aid their eventual identification, we evaluated cell types in which non-MHC Idd resistance genes in NOR mice exert disease-protective effects., Research Design and Methods: Adoptive transfer and bone marrow chimerism approaches tested the diabetogenic activity of CD4 and CD8 T-cells from NOR mice and NOD stocks congenic for NOR-derived Idd resistance loci. Tetramer staining and mimotope stimulation tested the frequency and proliferative capacity of CD4 BDC2.5-like cells. Regulatory T-cells (Tregs) were identified by Foxp3 staining and functionally assessed by in vitro suppression assays., Results: NOR CD4 T-cells were less diabetogenic than those from NOD mice. The failure of NOR CD4 T-cells to induce type 1 diabetes was not due to decreased proliferative capacity of BDC2.5 clonotypic-like cells. The frequency and function of Tregs in NOD and NOR mice were also equivalent. However, bone marrow chimerism experiments demonstrated that intrinsic factors inhibited the pathogenic activity of NOR CD4 T-cells. The NOR Idd9/11 resistance region on chromosome 4 was found to diminish the diabetogenic activity of CD4 but not CD8 T-cells., Conclusions: In conclusion, we demonstrated that a gene(s) within the Idd9/11 region regulates the diabetogenic activity of CD4 T-cells.

The purpose of our study was to develop a tool for blocking the function of a specific leukocyte ecto-enzyme in vivo. ART2.2 is a toxin-related ecto-enzyme that transfers the ADP-ribose moiety from NAD onto other cell surface proteins. ART2.2 induces T cell death by activating the cytolytic P2x7 purinoceptor via ADP-ribosylation. Here, we report the generation of ART2.2-blocking single domain antibodies from an immunized llama. The variable domain of heavy-chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses. Their long CDR3 endows VHH domains with the extraordinary capacity to extend into and block molecular clefts. Following intravenous injection, the ART2.2-specific VHH domains effectively shut off the enzymatic and cytotoxic activities of ART2.2 in lymphatic organs. This blockade was highly specific (blocking ART2.2 but not the related enzymes ART1 or ART2.1), rapid (within 15 min after injection), and reversible (24 h after injection). Our findings constitute a proof of principle that opens up a new avenue for targeting leukocyte ecto-enzymes in vivo and that can serve as a model also for developing new antidotes against ADP-ribosylating toxins.

Extracellular NAD and ATP exert multiple, partially overlapping effects on immune cells. Catabolism of both nucleotides by extracellular enzymes keeps extracellular concentrations low under steady-state conditions and generates metabolites that are themselves signal transducers. ATP and its metabolites signal through purinergic P2 and P1 receptors, whereas extracellular NAD exerts its effects by serving as a substrate for ADP-ribosyltransferases (ARTs) and NAD glycohydrolases/ADPR cyclases like CD38 and CD157. Both nucleotides activate the P2X7 purinoceptor, although by different mechanisms and with different characteristics. While ATP activates P2X7 directly as a soluble ligand, activation via NAD occurs by ART-dependent ADP-ribosylation of cell surface proteins, providing an immobilised ligand. P2X7 activation by either route leads to phosphatidylserine exposure, shedding of CD62L, and ultimately to cell death. Activation by ATP requires high micromolar concentrations of nucleotide and is readily reversible, whereas NAD-dependent stimulation begins at low micromolar concentrations and is more stable. Under conditions of cell stress or inflammation, ATP and NAD are released into the extracellular space from intracellular stores by lytic and non-lytic mechanisms, and may serve as "danger signals" to alert the immune response to tissue damage. Since ART expression is limited to naïve/resting T cells, P2X7-mediated NAD-induced cell death (NICD) specifically targets this cell population. In inflamed tissue, NICD may inhibit bystander activation of unprimed T cells, reducing the risk of autoimmunity. In draining lymph nodes, NICD may eliminate regulatory T cells or provide space for the preferential expansion of primed cells, and thus help to augment an immune response.

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 beta 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.

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.

Penetrance of the complex of genes predisposing the nonobese diabetic (NOD) mouse to autoimmune diabetes is affected by the maternal environment. NOD.CBALs-Tyr(+)/Lt is an agouti-pigmented Chromosome 7 congenic stock of NOD/Lt mice produced as a resource for embryo transfer experiments to provide the necessary maternal factors and allow the easy identification of NOD (albino) embryo donor phenotype. CBcNO6/Lt, a recombinant congenic agouti stock already containing approximately 50% NOD genome, was used as the donor source of a wild-type CBA tyrosinase allele. When the incidence of diabetes was assessed after nine generations of backcrossing and one generation of sib-sib mating, significant reduction in diabetes development was observed. No difference in diabetes development was observed in Tyr/Tyr(c) heterozygotes, showing that protection was recessive. Analysis of diabetes progression in another NOD stock congenic for C57BL/6 alleles on Chromosome 7 linked to the glucose phosphate isomerase (Gpi1(b)) locus provided no protection, indicating that the diabetes resistance (Idd) gene was distal to 34 cM (D7Mit346). Approximately 5 cM of the distal congenic region overlaps a region from C57L previously associated with protection when homozygous. The delayed onset and reduced frequency of diabetes in the NOD.CBALs-Tyr(+)/Lt stock is an advantage when females of this stock are used as surrogate mothers in studies involving hysterectomy or embryo transfers. Indeed, a newly developed NOD embryonic stem (ES) cell line injected into NOD.CBALs- Tyr(+)/Lt blastocysts produced approximately 50% live-born mice, of which approximately 11% were chimeric. Presumably because of high genomic instability, no germline transmission was observed.

Cytotoxicity induced by protein ADP-ribosylation is a common theme of certain bacterial toxins and of the mammalian ectoenzyme ART2. Exposure of T cells to NAD, the substrate for ART2-catalyzed ADP-ribosylation, induces exposure of phosphatidylserine, uptake of propidium iodide, and fragmentation of DNA. ART2-specific antibodies raised by gene gun immunization block NAD-induced apoptosis. ART2 catalyzed ADP-ribosylation of cell membrane proteins induces formation of cytolytic membrane pores by activating the P2X7 purinoceptor. This alternative pathway to T cell apoptosis could be triggered upon the release of NAD from intracellular stores, for example, during inflammatory tissue damage.

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.

We describe a patient with histologically confirmed relapsing polychondritis, an episodic systemic disorder. Although the etiology is unknown and its pathogenesis is incompletely understood, there is evidence strongly suggesting immunologically mediated mechanisms. Enzyme linked immunosorbent assays, immunoblotting and cellular immune responses using lymphocyte proliferation assays showed strong parallel humoral and cellular immune reactivities against collagens type IX and XI. There was also a considerable response to collagen type II which, however, was less pronounced compared to collagen type IX and was directed to native epitopes. Our findings demonstrate a highly distinct immune response to minor matrix collagens in a destructive cartilage disease and thus strongly argue against nonspecific anticollagen immune reactions simply representing epiphenomena resulting from cartilage damage.