NKG2D receptor signaling shapes T cell thymic education.

The role of natural killer group 2D (NKG2D) in peripheral T cells as a costimulatory receptor is well established. However, its contribution to T cell thymic education and functional imprint is unknown. Here, we report significant changes in development, receptor signaling, transcriptional program, and function in T cells from mice lacking NKG2D signaling. In C57BL/6 (B6) and OT-I mice, we found that NKG2D deficiency results in Vß chain usage changes and stagnation of the double-positive stage in thymic T cell development. We found that the expression of CD5 and CD45 in thymocytes from NKG2D deficient mice were reduced, indicating a direct influence of NKG2D on the strength of T cell receptor (TCR) signaling during the developmental stage of T cells. Depicting the functional consequences of NKG2D, peripheral OT-I NKG2D-deficient cells were unresponsive to ovalbumin peptide stimulation. Paradoxically, while αCD3/CD28 agonist antibodies led to phenotypic T cell activation, their ability to produce cytokines remained severely compromised. We found that OT-I NKG2D-deficient cells activate STAT5 in response to interleukin-15 but were unable to phosphorylate ERK or S6 upon TCR engagement, underpinning a defect in TCR signaling. Finally, we showed that NKG2D is expressed in mouse and human thymic T cells at the double-negative stage, suggesting an evolutionarily conserved function during T cell development. The data presented in this study indicate that NKG2D impacts thymic T cell development at a fundamental level by reducing the TCR threshold and affecting the functional imprint of the thymic progeny. In summary, understanding the impact of NKG2D on thymic T cell development and TCR signaling contributes to our knowledge of immune system regulation, immune dysregulation, and the design of immunotherapies.

Memory Precursors and Short-Lived Effector T cell Subsets Have Different Sensitivities to TGFß.

After exposure to an antigen, CD8 T cells reach a decision point about their fate: to become either short-lived effector cells (SLECs) or memory progenitor effector cells (MPECs). SLECs are specialized in providing an immediate effector function but have a shorter lifespan and lower proliferative capacity compared to MPECs. Upon encountering the cognate antigen during an infection, CD8 T cells rapidly expand and then contract to a level that is maintained for the memory phase after the peak of the response. Studies have shown that the contraction phase is mediated by TGFß and selectively targets SLECs, while sparing MPECs. The aim of this study is to investigate how the CD8 T cell precursor stage determines TGFß sensitivity. Our results demonstrate that MPECs and SLECs have differential responses to TGFß, with SLECs being more sensitive to TGFß than MPECs. This difference in sensitivity is associated with the levels of TGFßRI and RGS3, and the SLEC-related transcriptional activator T-bet binding to the TGFßRI promoter may provide a molecular basis for increased TGFß sensitivity in SLECs.

Inhibition of Histone Deacetylase (HDAC) Enhances Checkpoint Blockade Efficacy by Rendering Bladder Cancer Cells Visible for T Cell-Mediated Destruction.

Inhibitory checkpoint blockade therapy is an immunomodulatory strategy that results in the restoration of T cell functions, and its efficacy depends on the recognition of tumor cells for destruction. Considering the factors at play, one could propose that anti-tumor responses will not occur if tumor cells are immunologically invisible to T cells. In this study, we tested a strategy based on the modulation of cancer cell's immunovisibility through HDAC inhibition. In a model (heterotopic and orthotopic) of mouse urothelial bladder cancer, we demonstrated that the use of intratumoral or intravesical HDACi in combination with systemic anti-PD-1 was effective at inducing curative responses with durable anti-tumor immunity capable of preventing tumor growth at a distal site. Mechanistically, we determined that protective responses were dependent on CD8 cells, but not NK cells. Of significance, in an in vitro human model, we found that fully activated T cells fail at killing bladder cancer cells unless tumor cells were pretreated with HDACi. Complementary to this observation, we found that HDACi cause gene deregulation, that results in the upregulation of genes responsible for mediating immunorecognition, NKG2D ligands and HSP70. Taken together, these data indicate that HDAC inhibition results in the elimination of the tumor cell's "invisibility cloak" that prevents T cells from recognizing and killing them. Finally, as checkpoint blockade therapy moves into the adjuvant setting, its combined use with locally administrated HDACi represents a new approach to be included in our current therapeutic treatment toolbox.

NKG2D signaling certifies effector CD8 T cells for memory formation.

BACKGROUND: The development of memory responses is an evolutionary function of the adaptive immune system. We propose that for the immune system to populate the memory compartment with the best-suited CD8 T cells it utilizes a process of certification or molecular accreditation mediated through Natural Killer Group 2D (NKG2D). This process of certification assures that the memory compartment is filled with CD8 T cells that have demonstrated their ability to kill their cognate targets through a two-step process that utilizes T cell receptor (TCR) and NKG2D signaling. METHODS: One week after immunization with peptide-pulsed dendritic cells, NKG2D signaling was transiently blocked in vivo with a single injection of neutralizing antibodies. Under such conditions, we determined the importance of NKG2D signaling during the effector phase for memory formation without compromising NKG2D signaling at the memory phase. Both open (polyclonal) and closed (monoclonal) CD8 T cell repertoires were studied. RESULTS: We show that signaling through NKG2D mediated this certification. Temporary blockade of NKG2D signaling during the effector phase resulted in the formation of highly defective memory CD8 T cells characterized by altered expression of the ribosomal protein S6 and epigenetic modifiers, suggesting modifications in the T cell translational machinery and epigenetic programming. Finally, these uncertified memory cells were not protective against a B16 tumor challenge. CONCLUSION: Signaling through NKG2D during the effector phase (certification) favors the development of functional memory CD8 T cells, a previously undescribed role for NKG2D. Temporary blockade of NKG2D signaling during the effector phase results in the formation of highly defective memory CD8 T cells potentially by affecting the expression of the ribosomal protein S6 and epigenetic modifiers, suggesting alterations in T cell translational machinery and epigenetic programming.

Functions of NKG2D in CD8+ T cells: an opportunity for immunotherapy.

Natural killer group 2 member D (NKG2D) is a type II transmembrane receptor. NKG2D is present on NK cells in both mice and humans, whereas it is constitutively expressed on CD8+ T cells in humans but only expressed upon T-cell activation in mice. NKG2D is a promiscuous receptor that recognizes stress-induced surface ligands. In NK cells, NKG2D signaling is sufficient to unleash the killing response; in CD8+ T cells, this requires concurrent activation of the T-cell receptor (TCR). In this case, the function of NKG2D is to authenticate the recognition of a stressed target and enhance TCR signaling. CD28 has been established as an archetype provider of costimulation during T-cell priming. It has become apparent, however, that signals from other costimulatory receptors, such as NKG2D, are required for optimal T-cell function outside the priming phase. This review will focus on the similarities and differences between NKG2D and CD28; less well-described characteristics of NKG2D, such as the potential role of NKG2D in CD8+ T-cell memory formation, cancer immunity and autoimmunity; and the opportunities for targeting NKG2D in immunotherapy.

Bromide control, bifurcation and activation in the Belousov-Zhabotinsky reaction.

The unstirred, ferroin (Fe(phen)3(2+)) catalyzed Belousov-Zhabotinsky (BZ) reaction is the prototype oscillatory chemical system. Reaction media with added Br(-) appear red (reduced, low [Fe(phen)3(3+)]) during an induction period of several minutes, followed by the "spontaneous" formation of "pacemaker" sites, which oscillate between a blue, oxidized state (high [Fe(phen)3(3+)]) and the red, reduced state and generate target patterns of concentric, outwardly moving waves of oxidation (blue). Auto-oscillatory behavior is also seen in the Oregonator model of Field, Koros and Noyes (FKN), a robust, reduced model that captures qualitative BZ kinetics in the auto-oscillatory regime. However, the Oregonator model predicts a blue (oxidized) induction phase. Here we develop a generalized Oregonator-like model with no explicit bifurcation parameter that yields the observed transition from a red initial state to oscillatory dynamics, and displays a new bifurcation mechanism not seen in the original Oregonator.

Acoustic startle amplitude predicts vulnerability to develop post-traumatic stress hyper-responsivity and associated plasma corticosterone changes in rats.

Following exposure to trauma, a vulnerable sub-population of individuals develops post-traumatic stress disorder (PTSD) with characteristic persistent autonomic hyper-responsivity, associated increased startle response, and commonly altered hypothalamo-pituitary-adrenal regulation. A goal of this investigation was to identify a predictive marker for this vulnerability. Previous investigators have developed a model for PTSD in which male mice were exposed to a single brief episode of inescapable footshock followed by 1-min contextual reminders of this trauma at weekly intervals for 6 weeks. Exposure to these reminders induced a progressive and persistent increase in the amplitude of acoustic startle consistent with the persistently increased acoustic startle of individuals exhibiting PTSD. We adapted this model to adult male Wistar rats, with added characterization of initial (pre-trauma) startle response. After one episode of inescapable footshock (10 s, 2 mA) or control treatment followed by six weekly 1-min contextual reminders, acoustic startle was re-tested. Data were analyzed after dividing rats within each treatment into LOW vs MID vs HIGH (33% in each group) pre-treatment startle responders. Rats which exhibited pre-treatment LOW- and MID-range acoustic startle responses did not develop increased acoustic startle responses following subsequent traumatic stress+reminders ([TS+R]) treatment. However, rats which exhibited HIGH pre-treatment startle responses exhibited further significant (p<0.01) [TS+R]-induced persistent enhancement of this already elevated startle response. Furthermore, rats exhibiting HIGH pre-treatment startle responses were also the only subgroup which exhibited increased basal plasma corticosterone levels following [TS+R] treatment. These results suggest that initial pre-stress acoustic startle response can identify subgroups of rats which are predisposed to, or resistant to, developing a PTSD-like syndrome following subsequent trauma.

Olanzapine-induced weight gain and increased visceral adiposity is blocked by melatonin replacement therapy in rats.

The atypical antipsychotic drug olanzapine increases body weight and visceral adiposity in schizophrenia. In rats, aging-associated increased body weight and visceral adiposity are reversed by administration of the pineal hormone melatonin. We asked if melatonin similarly would reverse olanzapine-induced increased weight and visceral adiposity in rats. Four groups (n=11/group) of female rats (240-250 g) were treated for 8 weeks with olanzapine, melatonin, olanzapine+melatonin, or vehicle alone in drinking water. Body weight and food and water consumption were determined weekly, locomotor activity at weeks 3 and 6, and nocturnal plasma melatonin concentration at week 7. At week 8, the rats were killed and visceral (perirenal, retroperitoneal, omental, and mesenteric) fat pads dissected and weighed. Olanzapine treatment reduced nocturnal plasma melatonin by 55% (p<0.001), which was restored to control levels by olanzapine+melatonin. Body weight increased 18% in rats treated with olanzapine alone, but only 10% with olanzapine+melatonin, 5% with melatonin alone, and 7% with vehicle control. Body weight and visceral fat pad weight increases in rats treated with olanzapine alone were greater than in each of the other three groups (all p<0.01), which were not significantly different. These results suggest that olanzapine-induced increases in body weight and visceral adiposity may be at least in part secondary to olanzapine-induced reduction of plasma melatonin levels, and that melatonin may be useful for the management of olanzapine-induced weight gain in humans.

Dynorphin-mediated antinociceptive effects of L-arginine and SIN-1 (an NO donor) in mice.

The antinociceptive response of mice to the amino acid L-arginine (L-ARG) has been attributed to either an opioid mechanism or a non-opioid but nitric oxide (NO)-dependent mechanism. Earlier it was reported that the mechanism of nitrous oxide-induced antinociception involved opioid components and was also dependent on brain NO. This study was designed to determine whether the antinociceptive effects of L-ARG and the NO donor 3-morpholinosydnoimine (SIN-1) might be mediated by brain mechanisms similar to those that are responsible for nitrous oxide (N(2)O) antinociception. L-ARG and SIN-1 were administered to mice intracerebroventricularly (i.c.v.), and antinociception was assessed using the acetic acid abdominal constriction test. Both L-ARG and SIN-1 caused dose-related antinociceptive effects that were blocked by naloxone and norbinaltorphimine. The antinociceptive effects of both SIN-1 and L-ARG were also blocked to a greater extent by i.c.v. administration of a rabbit antiserum against rat dynorphin 1-13 than an antiserum against methionine-enkephalin, suggesting that the SIN-1 and L-ARG effects may be related to stimulated release of dynorphin. The antinociceptive effect of L-ARG was antagonized by an inhibitor of neuoronal NO synthase enzyme, indicating that L-ARG had to be converted to NO for its antinociceptive action. These findings indicate that the mechanisms of antinociceptive action of L-ARG and SIN-1 are both mediated by dynorphin and dependent on NO.