Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET:  Dopamine Movies of Cigarette Smoking

We describe experimental and statistical steps for creating dopamine movies of the brain from dynamic PET data. The movies represent minute-to-minute ...

Yale University

The immune response plays an important role in staving off cancer; however, mechanisms of immunosuppression hinder productive anti-tumor immunity. T cell dysfunction or exhaustion in tumor-bearing hosts is one such mechanism. PD-1 has been identified as a marker of exhausted T cells in chronic disease states, and blockade of PD-1-PD-1L interactions has been shown to partially restore T cell function. We have found that T cell immunoglobulin mucin (Tim) 3 is expressed on CD8(+) tumor-infiltrating lymphocytes (TILs) in mice bearing solid tumors. All Tim-3(+) TILs coexpress PD-1, and Tim-3(+)PD-1(+) TILs represent the predominant fraction of T cells infiltrating tumors. Tim-3(+)PD-1(+) TILs exhibit the most severe exhausted phenotype as defined by failure to proliferate and produce IL-2, TNF, and IFN-Î³. We further find that combined targeting of the Tim-3 and PD-1 pathways is more effective in controlling tumor growth than targeting either pathway alone.

Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity.

Microdialysis studies report that systemic alcohol increases extracellular dopamine (DA) in the rat striatum. The present study examined whether changes in striatal DA could be detected in rats using small animal positron emission tomography (PET). PET images were acquired in 44 alcohol-naÃ¯ve male Wistar and alcohol-preferring (P) rats. Subjects received up to three [(11) C]raclopride scans (rest, alcohol, and saline). Animals were anesthetized with isoflurane and secured on a stereotactic-like holder during all scans. Blood samples were collected from the tail or lateral saphenous vein of 12 animals 10 min after tracer injection for determination of blood alcohol concentration (BAC). Time activity curves were extracted from the striatum and the cerebellum and binding potential (BP(ND) ) was calculated as a measure of D(2) receptor availability. Wistars given 1.0 g kg(-1) alcohol (20%v/v) i.v. or 3.0 g kg(-1) alcohol (20%v/v) i.p. showed significant alcohol-induced decreases in BP(ND) . In P rats (given 1.5, 2.25, or 3.0 g kg(-1) alcohol), no individual group showed a statistical effect of alcohol on BP(ND) , but taken together, all P rats receiving i.p. alcohol had significantly lower BP(ND) than rest or saline scans. Large decreases in BP(ND) were primarily observed in rats with BAC above 200 mg%. Also, a significant difference was found between baseline BP(ND) of Wistars who had undergone jugular catheterization surgery for i.v. alcohol administration and those who had not. Preliminary results suggest that alcohol-induced DA release in the rat striatum is detectable using small animal PET given sufficiently large cohorts and adequate blood alcohol levels.

Imaging of alcohol-induced dopamine release in rats:preliminary findings with [(11) C]raclopride PET.

Type 1 diabetes (T1D) is an autoimmune disease that shows familial aggregation in humans and likely has genetic determinants. Disease linkage studies have revealed many susceptibility loci for T1D in mice and humans. The mouse T1D susceptibility locus insulin-dependent diabetes susceptibility 3 (Idd3), which has a homologous genetic interval in humans, encodes cytokine genes Il2 and Il21 and regulates diabetes and other autoimmune diseases; however, the cellular and molecular mechanisms of this regulation are still being elucidated. Here we show that T cells from NOD mice produce more Il21 and less Il2 and exhibit enhanced Th17 cell generation compared with T cells from NOD.Idd3 congenic mice, which carry the protective Idd3 allele from a diabetes-resistant mouse strain. Further, APCs from NOD and NOD.Idd3 mice played a central role in this differential Th17 cell development, and IL-21 signaling in APCs was pivotal to this process. Specifically, NOD-derived APCs showed increased production of pro-Th17 mediators and dysregulation of the retinoic acid (RA) signaling pathway compared with APCs from NOD.Idd3 and NOD.Il21r-deficient mice. These data suggest that the protective effect of the Idd3 locus is due, in part, to differential RA signaling in APCs and that IL-21 likely plays a role in this process. Thus, we believe APCs provide a new candidate for therapeutic intervention in autoimmune diseases.

Differential IL-21 signaling in APCs leads to disparate Th17 differentiation in diabetes-susceptible NOD and diabetes-resistant NOD.Idd3 mice.

Ectopic lymphoid follicles are hallmarks of chronic autoimmune inflammatory diseases such as multiple sclerosis (MS), rheumatoid arthritis, SjÃ¶gren's syndrome, and myasthenia gravis. However, the effector cells and mechanisms that induce their development are unknown. Here we showed that in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, Th17 cells specifically induced ectopic lymphoid follicles in the central nervous system (CNS). Development of ectopic lymphoid follicles was partly dependent on the cytokine interleukin 17 (IL-17) and on the cell surface molecule Podoplanin (Pdp), which was expressed on Th17 cells, but not on other effector T cell subsets. Pdp was also crucial for the development of secondary lymphoid structures: Pdp-deficient mice lacked peripheral lymph nodes and had a defect in forming normal lymphoid follicles and germinal centers in spleen and lymph node remnants. Thus, Th17 cells are uniquely endowed to induce tissue inflammation, characterized by ectopic lymphoid follicles within the target organ.

Th17 cells induce ectopic lymphoid follicles in central nervous system tissue inflammation.

Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis. In NOD mice, EAE develops as a relapsing-remitting disease that transitions to a chronic progressive disease, making the NOD model the only mouse model that recapitulates the full clinical disease course observed in most multiple sclerosis patients. We have generated a TCR transgenic mouse that expresses the Î±- and Î²-chains of a myelin oligodendrocyte glycoprotein (MOG) 35-55-reactive TCR (1C6) on the NOD background. 1C6 TCR transgenic mice spontaneously generate both CD4(+) and CD8(+) T cells that recognize MOG and produce proinflammatory cytokines, allowing for the first time to our knowledge the simultaneous examination of myelin-reactive CD4(+) and CD8(+) T cells in the same host. 1C6 CD8(+) T cells alone can induce optic neuritis and mild EAE with delayed onset; however, 1C6 CD4(+) T cells alone induce severe EAE and predominate in driving disease when both cell types are present. When 1C6 mice are crossed with mice bearing an IgH specific for MOG, the mice develop spontaneous EAE with high incidence, but surprisingly the disease pattern does not resemble the neuromyelitis optica-like disease observed in mice bearing CD4(+) T cells and B cells reactive to MOG on the C57BL/6 background. Collectively, our data show that although myelin-reactive CD8(+) T cells contribute to disease, disease is primarily driven by myelin-reactive CD4(+) T cells and that the coexistence of myelin-reactive T and B cells does not necessarily result in a distinct pathological phenotype.

A transgenic model of central nervous system autoimmunity mediated by CD4+ and CD8+ T and B cells.

A custom designed markerless tracking system was demonstrated to be applicable for positron emission tomography (PET) brain imaging. Precise head motion registration is crucial for accurate motion correction (MC) in PET imaging. State-of-the-art tracking systems applied with PET brain imaging rely on markers attached to the patient's head. The marker attachment is the main weakness of these systems. A healthy volunteer participating in a cigarette smoking study to image dopamine release was scanned twice for 2 h with (11)C-racolopride on the high resolution research tomograph (HRRT) PET scanner. Head motion was independently measured, with a commercial marker-based device and the proposed vision-based system. A list-mode event-by-event reconstruction algorithm using the detected motion was applied. A phantom study with hand-controlled continuous random motion was obtained. Motion was time-varying with long drift motions of up to 18 mm and regular step-wise motion of 1-6 mm. The evaluated measures were significantly better for motion-corrected images compared to no MC. The demonstrated system agreed with a commercial integrated system. Motion-corrected images were improved in contrast recovery of small structures.

List-mode PET motion correction using markerless head tracking: proof-of-concept with scans of human subject.

[(18)F]FPEB is a positron emission tomography tracer which, in preclinical studies, has shown high specificity and selectivity toward the metabotropic glutamate receptor 5 (mGluR5). It possesses the potential to be used in human studies to evaluate mGluR5 function in a range of neuropsychiatric disorders, such as anxiety and Fragile X syndrome. To define optimal scan methodology, healthy human subjects were scanned for 6 hours following either a bolus injection (n=5) or bolus-plus-constant-infusion (n=5) of [(18)F]FPEB. Arterial blood samples were collected and parent fraction measured by high-performance liquid chromatography (HPLC) to determine the metabolite-corrected plasma input function. Time activity curves were extracted from 13 regions and fitted by various models to estimate V(T) and BPND. [(18)F]FPEB was well fitted by the two-tissue compartment model, MA1 (t*=30), and MRTM (using cerebellum white matter as a reference). Highest V(T) values were observed in the anterior cingulate and caudate, and lowest V(T) values were observed in the cerebellum and pallidum. For kinetic modeling studies, VT and BPND were estimated from bolus or bolus-plus-constant-infusion scans as short as 90 minutes. Bolus-plus-constant-infusion of [(18)F]FPEB reduced intersubject variability in V(T) and allowed equilibrium analysis to be completed with a 30-minute scan, acquired 90-120 minutes after the start of injection.

Kinetic analysis of the metabotropic glutamate subtype 5 tracer [(18)F]FPEB in bolus and bolus-plus-constant-infusion studies in humans.

Conventional PET methods to estimate [(11)C]raclopride binding potential (BP ND) assume that endogenous dopamine concentration does not change during the scan time. However, this assumption is purposely violated in studies using pharmacological or behavioral stimuli to invoke acute dopamine release. When the assumption of steady-state dopamine is violated, conventional analysis methods may produce biased or even unusable estimates of BP ND. To illustrate this problem, we examined the effect of scan duration on Î”BP ND estimated by three common analysis methods (simplified reference tissue model, Logan graphical reference method, and equilibrium analysis) applied to simulated and experimental single-scan activation studies. The activation - dopamine release - in both the simulated and experimental studies was brief. Simulations showed Î”BP ND to be highly dependent on the window of data used to determine BP ND in the activation state. A similar pattern was seen in the data from human smoking studies. No such pattern of Î”BP ND dependence on the window of data used was apparent in simulations where dopamine was held constant. The dependence of Î”BP ND on the duration of data analyzed illustrates the inability of conventional methods to reliably quantify short-lived increases in endogenous dopamine.

Limitations of SRTM, Logan graphical method, and equilibrium analysis for measuring transient dopamine release with [(11)C]raclopride PET.

T-cell immunoglobulin mucin 3 (TIM3) is an inhibitory molecule that has emerged as a key regulator of dysfunctional or exhausted CD8(+) T cells arising in chronic diseases such as cancer. In addition to exhausted CD8(+) T cells, highly suppressive regulatory T cells (Tregs) represent a significant barrier against the induction of antitumor immunity. We have found that the majority of intratumoral FOXP3(+) Tregs express TIM3. TIM3(+) Tregs co-express PD-1, are highly suppressive and comprise a specialized subset of tissue Tregs that are rarely observed in the peripheral tissues or blood of tumor-bearing mice. The co-blockade of the TIM3 and PD-1 signaling pathways in vivo results in the downregulation of molecules associated with TIM3(+) Treg suppressor functions. This suggests that the potent clinical efficacy of co-blocking TIM3 and PD-1 signal transduction cascades likely stems from the reversal of T-cell exhaustion combined with the inhibition of regulatory T-cell function in tumor tissues. Interestingly, we find that TIM3(+) Tregs accumulate in the tumor tissue prior to the appearance of exhausted CD8(+) T cells, and that the depletion of Tregs at this stage interferes with the development of the exhausted phenotype by CD8(+) T cells. Collectively, our data indicate that TIM3 marks highly suppressive tissue-resident Tregs that play an important role in shaping the antitumor immune response in situ, increasing the value of TIM3-targeting therapeutic strategies against cancer.

TIM3(+)FOXP3(+) regulatory T cells are tissue-specific promoters of T-cell dysfunction in cancer.

La mucine de lymphocytes immunoglobuline 3 (TIM3) est une mol&eacute;cule inhibitrice qui a &eacute;merg&eacute; comme un r&eacute;gulateur cl&eacute; de cellules T CD8 dysfonctionnelles ou &eacute;puis&eacute;s d&eacute;coulant des maladies chroniques comme le cancer. En plus des cellules T CD8 &eacute;puis&eacute;s, les lymphocytes T r&eacute;gulateurs hautement suppressives (Tregs) repr&eacute;sentent un obstacle important contre l&#39;induction d&#39;une immunit&eacute; antitumorale. Nous avons constat&eacute; que la majorit&eacute; des intratumorale FOXP3(+) Tregs express TIM3. TIM3(+) Tregs co-express PD-1, sont hautement suppressives et forment un sous-ensemble sp&eacute;cialis&eacute; de tissu Tregs qui sont rarement observ&eacute;s dans les tissus p&eacute;riph&eacute;riques ou le sang des souris de tumeur-roulement. Le co-blockade des voies de signalisation TIM3 et PD-1 in vivo se traduit par la diminution de l&#39;expression de mol&eacute;cules associ&eacute;es aux fonctions de suppresseur de Treg TIM3(+). Cela donne &agrave; penser que l&#39;efficacit&eacute; clinique puissante de co-blocking TIM3 et PD-1 signal transduction cascades probable provient de l&#39;inversion de l&#39;&eacute;puisement de lymphocytes combin&eacute;e &agrave; l&#39;inhibition de la fonction r&eacute;gulatrice de lymphocytes T dans les tissus de la tumeur. Fait int&eacute;ressant, nous trouvons que TIM3(+) Tregs s&#39;accumulent dans le tissu de la tumeur avant l&#39;apparition de cellules de T de CD8 &eacute;puis&eacute;s, et que l&#39;&eacute;puisement des Tregs &agrave; ce stade interf&egrave;re avec le d&eacute;veloppement du ph&eacute;notype &eacute;puis&eacute; par les cellules T CD8. Collectivement, nos donn&eacute;es indiquent que TIM3 marque hautement suppressive Tregs tissu-r&eacute;sident qui jouent un r&ocirc;le important dans l&#39;&eacute;laboration de la r&eacute;ponse immunitaire anti-tumorale in situe, valorisation des TIM3-ciblage des strat&eacute;gies th&eacute;rapeutiques contre le cancer.

Jenna M. Sullivan

Diagnostic Radiology,
Yale PET Center,
Biomedical Engineering

Yale University

Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking

Jenna M. Sullivan

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Diagnostic Radiology,Yale PET Center,Biomedical Engineering

Yale University

Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking

Diagnostic Radiology,
Yale PET Center,
Biomedical Engineering