Ectopic lymphoid follicles are hallmarks of chronic autoimmune inflammatory diseases such as multiple sclerosis (MS), rheumatoid arthritis, Sjögren'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 but not other T cell subsets induce ectopic follicles in the CNS ► Th17 cells but not other T cell subsets express Podoplanin ► Formation of ectopic follicles depends on IL-17 and on Podoplanin ► Podoplanin is also crucial for formation of organized secondary lymphoid structures
Cigarette smoking is a major public health danger. Women and men smoke for different reasons and cessation treatments, such as the nicotine patch, are preferentially beneficial to men. The biological ...substrates of these sex differences are unknown. Earlier PET studies reported conflicting findings but were each hampered by experimental and/or analytical limitations. Our new image analysis technique, lp-ntPET (Normandin et al., 2012; Morris et al., 2013; Kim et al., 2014), has been optimized for capturing brief (lasting only minutes) and highly localized dopaminergic events in dynamic PET data. We coupled our analysis technique with high-resolution brain scanning and high-frequency motion correction to create the optimal experiment for capturing and characterizing the effects of smoking on the mesolimbic dopamine system in humans. Our main finding is that male smokers smoking in the PET scanner activate dopamine in the right ventral striatum during smoking but female smokers do not. This finding-men activating more ventrally than women-is consistent with the established notion that men smoke for the reinforcing drug effect of cigarettes whereas women smoke for other reasons, such as mood regulation and cue reactivity. lp-ntPET analysis produces a novel multidimensional endpoint: voxel-level temporal patterns of neurotransmitter release ("DA movies") in individual subjects. By examining these endpoints quantitatively, we demonstrate that the timing of dopaminergic responses to cigarette smoking differs between men and women. Men respond consistently and rapidly in the ventral striatum whereas women respond faster in a discrete subregion of the dorsal putamen.
Although there are no conventional lymphatic vessels in the brain, fluid and solutes drain along basement membranes (BMs) of cerebral capillaries and arteries towards the subarachnoid space and ...cervical lymph nodes. Convective influx/glymphatic entry of the cerebrospinal fluid (CSF) into the brain parenchyma occurs along the pial-glial BMs of arteries. This project tested the hypotheses that pial-glial BM of arteries are thicker in the midbrain, allowing more glymphatic entry of CSF. The
MRI and PET images were obtained from a 4.2-year-old dog, whereas the post-mortem electron microscopy was performed in a 12-year-old dog. We demonstrated a significant increase in the thickness of the pial-glial BM in the midbrain compared with the same BM in different regions of the brain and an increase in the convective influx of fluid from the subarachnoid space. These results are highly significant for the intrathecal drug delivery into the brain, indicating that the midbrain is better equipped for convective influx/glymphatic entry of the CSF.
The intrathecal (IT) dosing route introduces drugs directly into the CSF to bypass the blood-brain barrier and gain direct access to the CNS. We evaluated the use of convective forces acting on the ...cerebrospinal fluid as a means for increasing rostral delivery of IT dosed radioactive tracer molecules and antisense oligonucleotides (ASO) in the monkey CNS. We also measured the cerebral spinal fluid (CSF) volume in a group of cynomolgus monkeys.
There are three studies presented, in each of which cynomolgus monkeys were injected into the IT space with radioactive tracer molecules and/or ASO by lumbar puncture in either a low or high volume. The first study used the radioactive tracer
Cu-DOTA and PET imaging to evaluate the effect of the convective forces. The second study combined the injection of the radioactive tracer
Tc-DTPA and ASO, then used SPECT imaging and ex vivo tissue analysis of the effects of convective forces to bridge between the tracer and the ASO distributions. The third experiment evaluated the effects of different injection volumes on the distribution of an ASO. In the course of performing these studies we also measured the CSF volume in the subject monkeys by Magnetic Resonance Imaging.
It was consistently found that larger bolus dose volumes produced greater rostral distribution along the neuraxis. Thoracic percussive treatment also increased rostral distribution of low volume injections. There was little added benefit on distribution by combining the thoracic percussive treatment with the high-volume injection. The CSF volume of the monkeys was found to be 11.9 ± 1.6 cm
.
These results indicate that increasing convective forces after IT injection increases distribution of molecules up the neuraxis. In particular, the use of high IT injection volumes will be useful to increase rostral CNS distribution of therapeutic ASOs for CNS diseases in the clinic.
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-γ. 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.
Foxp3
regulatory T (T
) cells are phenotypically and functionally diverse and broadly distributed in lymphoid and nonlymphoid tissues. However, the pathways guiding the differentiation of ...tissue-resident T
cell populations have not been well defined. By regulating E-protein function, Id3 controls the differentiation of CD8
effector T cells and is essential for T
cell maintenance and function. We show that dynamic expression of Id3 helps define three distinct mouse T
cell populations: Id3
CD62L
CD44
central T
cells, Id3
CD62L
CD44
effector T
(eT
) cells, and Id3
eT
cells. Adoptive transfer experiments and transcriptome analyses support a stepwise model of differentiation from Id3
central T
to Id3
eT
to Id3
eT
cells. Furthermore, Id3
eT
cells have high expression of functional inhibitory markers and a transcriptional signature of tissue-resident T
cells. Accordingly, Id3
eT
cells are highly enriched in nonlymphoid organs but virtually absent from blood and lymph. Thus, we propose that tissue-resident T
cells develop in a multistep process associated with Id3 downregulation.
Abstract Activating mutations of the epidermal growth factor receptor (EGFR) occur in multiple tumor types, including non-small cell lung cancer (NSCLC) and malignant glioma, and have become targets ...for therapeutic intervention. The determination of EGFR mutation status using a noninvasive, molecular imaging approach has the potential for clinical utility. In this study, we investigated 11 C-erlotinib positron emission tomography (PET) imaging as a tool to identify activating mutations of EGFR in both glioma and NSCLC xenografts. Radiotracer specific binding was determined for high and low specific activity (SA) 11 C-erlotinib PET scans in mice bearing synchronous human cancer xenografts with different EGFR expression profiles (PC9, HCC827, U87, U87 ΔEGFR, and SW620). Although xenograft immunohistochemistry demonstrated constitutive EGFR phosphorylation, PET scan analysis using the Simplified Reference Tissue Model showed that only kinase domain mutant NSCLC (HCC827 and PC9) had significantly greater binding potentials in high versus low SA scans. Xenografts with undetectable EGFR expression (SW620), possessing wild-type EGFR (U87), and expressing an activating extracellular domain mutation (U87 ΔEGFR) were indistinguishable under both high and low SA scan conditions. The results suggest that 11 C-erlotinib is a promising radiotracer that could provide a novel clinical methodology for assessing EGFR and erlotinib interactions in patients with tumors that harbor EGFR-activating kinase domain mutations.
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.
CD8
T cells respond to signals via the T cell receptor (TCR), costimulatory molecules, and immunoregulatory cytokines by developing into diverse populations of effector and memory cells. The relative ...strength of phosphoinositide 3-kinase (PI3K) signaling early in the T cell response can dramatically influence downstream effector and memory T cell differentiation. We show that initial PI3K signaling during T cell activation results in up-regulation of the signaling scaffold B cell adaptor for PI3K (BCAP), which further potentiates PI3K signaling and promotes the accumulation of CD8
T cells with a terminally differentiated effector phenotype. Accordingly, BCAP-deficient CD8
T cells have attenuated clonal expansion and altered effector and memory T cell development following infection with
Thus, induction of BCAP serves as a positive feedback circuit to enhance PI3K signaling in activated CD8
T cells, thereby acting as a molecular checkpoint regulating effector and memory T cell development.
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.
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