18F-AraG PET for CD8 Profiling of Tumors and Assessment of Immunomodulation by Chemotherapy.

Most clinical trials exploring various combinations of chemo- and immunotherapy rely on serial biopsy to provide information on immune response. The aim of this study was to assess the value of 18F-arabinosyl guanine (18F-AraG) as a noninvasive tool that profiles tumors on the basis of the key player in adaptive antitumor response, CD8+ cells, and evaluates the immunomodulatory effects of chemotherapy. Methods: To evaluate the ability of 18F-AraG to report on the presence of CD8+ cells within the tumor microenvironment, we imaged a panel of syngeneic tumor models (MC38, CT26, LLC, A9F1, 4T1, and B16F10) and correlated the signal intensity with the number of lymphocytes found in the tumors. The capacity of 18F-AraG to detect immunomodulatory effects of chemotherapy was determined by longitudinal imaging of tumor-bearing mice (MC38 and A9F1) undergoing 2 types of chemotherapy: oxaliplatin/cyclophosphamide, shown to induce immunogenic cell death, and paclitaxel/carboplatin, reported to cause immunogenically silent tumor cell death. Results: In the tumor panel, 18F-AraG revealed strikingly different uptake patterns resembling cancer-immune phenotypes observed in the clinic. A statistically significant correlation was found between the 18F-AraG signal and the number of PD-1-positive CD8+ cells isolated from the tumors (r2 = 0.528, P < 0.0001). In the MC38 model, paclitaxel/carboplatin did not result in an appreciable change in signal after therapy (1.69 ± 0.25 vs. 1.50 ± 0.33 percentage injected dose per gram), but oxaliplatin/cyclophosphamide treatment led to close to a 2.4-fold higher 18F-AraG signal (1.20 ± 0.31 vs. 2.84 ± 0.93 percentage injected dose per gram). The statistically significant increase in signal after oxaliplatin/cyclophosphamide was also observed in the A9F1 model (0.95 ± 0.36 vs. 1.99 ± 0.54 percentage injected dose per gram). Conclusion: The ability of 18F-AraG PET to assess the location and function of CD8+ cells, as well immune activity within tumors after immune priming therapy, warrants further investigation into its utility for patient selection, evaluation of optimal time to deliver immunotherapies, and assessment of combinatorial therapies.

Imaging of Activated T Cells as an Early Predictor of Immune Response to Anti-PD-1 Therapy.

Compelling evidence points to immune cell infiltration as a critical component of successful immunotherapy. However, there are currently no clinically available, noninvasive methods capable of evaluating immune contexture prior to or during immunotherapy. In this study, we evaluate a T-cell-specific PET agent, [18F]F-AraG, as an imaging biomarker predictive of response to checkpoint inhibitor therapy. We determined the specificity of the tracer for activated T cells in vitro and in a virally induced model of rhabdomyosarcoma. Of all immune cells tested, activated human CD8+ effector cells showed the highest accumulation of [18F]F-AraG. Isolation of lymphocytes from the rhabdomyosarcoma tumors showed that more than 80% of the intratumoral signal came from accumulation of [18F]F-AraG in immune cells, primarily CD8+ and CD4+. Longitudinal monitoring of MC38 tumor-bearing mice undergoing anti-PD-1 treatment revealed differences in signal between PD-1 and isotype antibody-treated mice early into treatment. The differences in [18F]F-AraG signal were also apparent between responders and nonresponders to anti-PD-1 therapy. Importantly, we found that the signal in the tumor-draining lymph nodes provides key information about response to anti-PD-1 therapy. Overall, [18F]F-AraG has potential to serve as a much needed immunomonitoring clinical tool for timely evaluation of immunotherapy. SIGNIFICANCE: These findings reveal differences in T-cell activation between responders and nonresponders early into anti-PD-1 treatment, which may impact many facets of immuno-oncology, including patient selection, management, and development of novel combinatorial approaches.

Aryl hydrocarbon receptor signaling regulates NF-κB RelB activation during dendritic-cell differentiation.

How the aryl hydrocarbon receptor (AhR) regulates dendritic-cell (DC) differentiation is unknown. We show that activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) caused enhanced differentiation from immature DCs (IDCs) to mature DCs (MDCs) in the bone-marrow-derived DCs (BMDC) from B6 wild-type mice but not in the BMDCs from AhR-null mice as indicated by the expression of CD11c and class II major histocompatibility complex (MHC). Enhanced maturation of BMDCs was associated with elevated levels of CD86 and an increased AhR-dependent nuclear accumulation of nuclear factor-kappa-light-chain enhancer of activated B cell (NF-κB) member RelB in BMDCs. The expression of interleukin (IL) 10 and chemokine DC-CK1 was suppressed, whereas that of CXCL2, CXCL3 and IL-22 was significantly increased in AhR-activated BMDCs. Furthermore, TCDD induced expression of the regulatory enzymes indoleamine 2,3-dioxygenase (IDO1) and indoleamine 2,3-dioxygenase-like 1 (IDO2). Increased expression of IDO2 was associated with coexpression of the cell-surface marker CCR6. Interestingly, mRNA expression of the chemokine receptor CCR6 was drastically decreased in AhR-null IDCs and MDCs. Overall, these data demonstrate that AhR modifies the maturation of BMDCs associated with the induction of the regulatory enzyme IDO and altered expression of cytokine, chemokines and DC-specific surface markers and receptors.

Mice expressing T4826I-RYR1 are viable but exhibit sex- and genotype-dependent susceptibility to malignant hyperthermia and muscle damage.

Mutation T4825I in the type 1 ryanodine receptor (RYR1(T4825I/+)) confers human malignant hyperthermia susceptibility (MHS). We report a knock-in mouse line that expresses the isogenetic mutation T4826I. Heterozygous RYR1(T4826I/+) (Het) or homozygous RYR1(T4826I/T4826I) (Hom) mice are fully viable under typical rearing conditions but exhibit genotype- and sex-dependent susceptibility to environmental conditions that trigger MH. Hom mice maintain higher core temperatures than WT in the home cage, have chronically elevated myoplasmic[Ca(2+)](rest), and present muscle damage in soleus with a strong sex bias. Mice subjected to heat stress in an enclosed 37°C chamber fail to trigger MH regardless of genotype, whereas heat stress at 41°C invariably triggers fulminant MH in Hom, but not Het, mice within 20 min. WT and Het female mice fail to maintain euthermic body temperature when placed atop a bed whose surface is 37°C during halothane anesthesia (1.75%) and have no hyperthermic response, whereas 100% Hom mice of either sex and 17% of the Het males develop fulminant MH. WT mice placed on a 41°C bed maintain body temperature while being administered halothane, and 40% of the Het females and 100% of the Het males develop fulminant MH within 40 min. Myopathic alterations in soleus were apparent by 12 mo, including abnormally distributed and enlarged mitochondria, deeply infolded sarcolemma, and frequent Z-line streaming regions, which were more severe in males. These data demonstrate that an MHS mutation within the S4-S5 cytoplasmic linker of RYR1 confers genotype- and sex-dependent susceptibility to pharmacological and environmental stressors that trigger fulminant MH and promote myopathy.

Aryl hydrocarbon receptor signaling mediates expression of indoleamine 2,3-dioxygenase.

Aryl hydrocarbon receptor (AhR) activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to immune suppression associated with the induction of regulatory T cells (T(reg)) expressing the transcription factor Foxp3. The immunological mechanisms of suppression are not well understood however dendritic cells (DC) are considered a key target for AhR-mediated immune suppression. Here we show that activation of AhR by TCDD induces DC indoleamine 2,3-dioxygenase 1 (IDO1) and indoleamine 2,3-dioxygenase-like protein (IDO2). Induction of IDO1 and IDO2 was also found in lung and spleen associated with an increase of the T(reg) marker Foxp3 in spleen of TCDD-treated C57BL/6 mice, which is suppressed by inhibition of IDO. These data indicate that AhR-activation is an important signaling pathway for IDO expression and suggest a critical role of IDO in the mechanism leading to the generation of T(reg) that mediates the immune suppression through activation of AhR.

Pharmacologic and functional characterization of malignant hyperthermia in the R163C RyR1 knock-in mouse.

BACKGROUND: Malignant hyperthermia is a pharmacogenetic disorder affecting humans, dogs, pigs, and horses. In the majority of human cases and all cases in animals, malignant hyperthermia has been associated with missense mutations in the skeletal ryanodine receptor (RyR1). METHODS: The authors used a "knock-in" targeting vector to create mice carrying the RyR1 R163C malignant hyperthermia mutation. RESULTS: Validation of this new mouse model of human malignant hyperthermia susceptibility includes (1) proof of transcription of the R163C allele and expression of ryanodine receptor protein in R163C heterozygous and R163C homozygous animals; (2) fulminant malignant hyperthermia episodes in R163C heterozygous mice after exposure to 1.25-1.75% halothane or an ambient temperature of 42 degrees C characterized by increased rectal temperature, respiratory rate, and inspiratory effort, with significant blood biochemical changes indicating metabolic acidosis, ending in death and hyperacute rigor mortis; (3) intraperitoneal pretreatment with dantrolene provided 100% protection from the halothane-triggered fulminant malignant hyperthermia episode; (4) significantly increased sensitivity (decreased effective concentration causing 50% of the maximal response) of R163C heterozygous and homozygous myotubes to caffeine, 4-chloro-m-cresol, and K-induced depolarization; (5) R163C heterozygous and homozygous myotubes have a significantly increased resting intracellular Ca concentration compared with wild type; (6) R163C heterozygous sarcoplasmic reticulum membranes have a twofold higher affinity (Kd = 35.4 nm) for [H]ryanodine binding compared with wild type (Kd = 80.1 nm) and a diminished inhibitory regulation by Mg. CONCLUSIONS: Heterozygous R163C mice represent a valid model for studying the mechanisms that cause the human malignant hyperthermia syndrome.

Uncoupling of ATP-mediated calcium signaling and dysregulated interleukin-6 secretion in dendritic cells by nanomolar thimerosal.

Dendritic cells (DCs) , a rare cell type widely distributed in the soma, are potent antigen-presenting cells that initiate primary immune responses. DCs rely on intracellular redox state and calcium (Ca2+) signals for proper development and function, but the relationship between these two signaling systems is unclear. Thimerosal (THI) is a mercurial used to preserve vaccines and consumer products, and is used experimentally to induce Ca2+ release from microsomal stores. We tested adenosine triphosphate (ATP) -mediated Ca2+ responses of DCs transiently exposed to nanomolar THI. Transcriptional and immunocytochemical analyses show that murine myeloid immature DCs (IDCs) and mature DCs (MDCs) express inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) Ca2+ channels, known targets of THI. IDCs express the RyR1 isoform in a punctate distribution that is densest near plasma membranes and within dendritic processes, whereas IP3Rs are more generally distributed. RyR1 positively and negatively regulates purinergic signaling because ryanodine (Ry) blockade a) recruited 80% more ATP responders, b) shortened ATP-mediated Ca2+ transients > 2-fold, and c) produced a delayed and persistent rise (>/= 2-fold) in baseline Ca2+. THI (100 nM, 5 min) recruited more ATP responders, shortened the ATP-mediated Ca2+ transient (>/= 1.4-fold) , and produced a delayed rise (>/= 3-fold) in the Ca2+ baseline, mimicking Ry. THI and Ry, in combination, produced additive effects leading to uncoupling of IP3R and RyR1 signals. THI altered ATP-mediated interleukin-6 secretion, initially enhancing the rate of cytokine secretion but suppressing cytokine secretion overall in DCs.DCs are exquisitely sensitive to THI, with one mechanism involving the uncoupling of positive and negative regulation of Ca2+ signals contributed by RyR1.

Oxygen tension regulates the in vitro maturation of GM-CSF expanded murine bone marrow dendritic cells by modulating class II MHC expression.

Conventional culture conditions for GM-CSF expanded murine bone marrow derived dendritic cells (BMDCs) uses ambient (hyperoxic) oxygen pressure (20% v/v, 152 Torr) and medium supplemented with the thiol 2-mercaptoethanol (2-Me). Given the redox activities of O2 and 2-Me, the effects of 2%, 5%, 10%, and 20% v/v O2 atmospheres and omitting 2-Me from the medium were tested upon the generation of GM-CSF expanded BMDCs. DC yield, phenotype and function were compared to BMDCs grown using conventional conditions. All cultures yielded DC subsets with CD11c+ MHC II(NEG), CD11c+ MHC II(INT), CD11c+ MHC II(HI) expression phenotypes, classed as precursor, immature, and mature DCs (IDC, MDC). Low O2 tensions generated significantly fewer precursor DCs, and more IDCs and MDCs. Cytometer sorted precursor DCs expressed surface class II MHC after transfer to low, but not high O2 atmospheres. Expression of myeloid markers was similar between BMDC cultures generated in 5% O2 or conventional conditions, and MDCs from low O2 cultures had the morphology typical of mature myeloid DCs. IDCs and MDCs from low O2 and conventional culture conditions were similarly potent allostimulatory APCs. The O2 tension (but not 2-Me addition) in vitro significantly influences overall DC subset frequencies and yield, and governs DC maturation by regulating the surface class II MHC expression of GM-CSF expanded BMDC cultures.