Immune homeostasis in tissues is achieved through a delicate balance between pathogenic T-cell responses directed at tissue-specific antigens and the ability of the tissue to inhibit these responses. ...The mechanisms by which tissues and the immune system communicate to establish and maintain immune homeostasis are currently unknown. Clinical evidence suggests that chronic or repeated exposure to self antigen within tissues leads to an attenuation of pathological autoimmune responses, possibly as a means to mitigate inflammatory damage and preserve function. Many human organ-specific autoimmune diseases are characterized by the initial presentation of the disease being the most severe, with subsequent flares being of lesser severity and duration. In fact, these diseases often spontaneously resolve, despite persistent tissue autoantigen expression. In the practice of antigen-specific immunotherapy, allergens or self antigens are repeatedly injected in the skin, with a diminution of the inflammatory response occurring after each successive exposure. Although these findings indicate that tissues acquire the ability to attenuate autoimmune reactions upon repeated responses to antigens, the mechanism by which this occurs is unknown. Here we show that upon expression of self antigen in a peripheral tissue, thymus-derived regulatory T cells (T(reg) cells) become activated, proliferate and differentiate into more potent suppressors, which mediate resolution of organ-specific autoimmunity in mice. After resolution of the inflammatory response, activated T(reg) cells are maintained in the target tissue and are primed to attenuate subsequent autoimmune reactions when antigen is re-expressed. Thus, T(reg) cells function to confer 'regulatory memory' to the target tissue. These findings provide a framework for understanding how T(reg) cells respond when exposed to self antigen in peripheral tissues and offer mechanistic insight into how tissues regulate autoimmunity.
Temporal control of protein levels in cells and living animals can be used to improve our understanding of protein function. In addition, control of engineered proteins could be used in therapeutic ...applications. PRoteolysis-TArgeting Chimeras (PROTACs) have emerged as a small-molecule-driven strategy to achieve rapid, post-translational regulation of protein abundance via recruitment of an E3 ligase to the target protein of interest. Here, we develop several PROTAC molecules by covalently linking the antibiotic trimethoprim (TMP) to pomalidomide, a ligand for the E3 ligase, Cereblon. These molecules induce degradation of proteins of interest (POIs) genetically fused to a small protein domain, E. coli dihydrofolate reductase (eDHFR), the molecular target of TMP. We show that various eDHFR-tagged proteins can be robustly degraded to 95% of maximum expression with PROTAC molecule 7c. Moreover, TMP-based PROTACs minimally affect the expression of immunomodulatory imide drug (IMiD)-sensitive neosubstrates using proteomic and biochemical assays. Finally, we show multiplexed regulation with another known degron-PROTAC pair, as well as reversible protein regulation in a rodent model of metastatic cancer, demonstrating the formidable strength of this system. Altogether, TMP PROTACs are a robust approach for selective and reversible degradation of eDHFR-tagged proteins in vitro and in vivo.
BACKGROUND. Several molecular imaging strategies can identify bacterial infections in humans. PET affords the potential for sensitive infection detection deep within the body. Among PET-based ...approaches, antibiotic-based radiotracers, which often target key bacterial-specific enzymes, have considerable promise. One question for antibiotic radiotracers is whether antimicrobial resistance (AMR) reduces specific accumulation within bacteria, diminishing the predictive value of the diagnostic test. METHODS. Using a PET radiotracer based on the antibiotic trimethoprim (TMP), 11C-TMP, we performed in vitro uptake studies in susceptible and drug-resistant bacterial strains and whole-genome sequencing (WGS) in selected strains to identify TMP resistance mechanisms. Next, we queried the NCBI database of annotated bacterial genomes for WT and resistant dihydrofolate reductase (DHFR) genes. Finally, we initiated a first-in-human protocol of "C-TMP in patients infected with both TMP-sensitive and TMP-resistant organisms to demonstrate the clinical feasibility of the tool. RESULTS. We observed robust "C-TMP uptake in our panel of TMP-sensitive and -resistant bacteria, noting relatively variable and decreased uptake in a few strains of P. aeruginosa and E. coli. WGS showed that the vast majority of clinically relevant bacteria harbor a WT copy of DHFR, targetable by "C-TMP, and that despite the AMR, these strains should be imageable. Clinical imaging of patients with C-TMP demonstrated focal radiotracer uptake in areas of infectious lesions. CONCLUSION. This work highlights an approach to imaging bacterial infection in patients, which could affect our understanding of bacterial pathogenesis as well as our ability to better diagnose infections and monitor response to therapy. TRIAL REGISTRATION. ClinicalTrials.gov NCT03424525. FUNDING. Institute for Translational Medicine and Therapeutics, Burroughs Wellcome Fund, NIH Office of the Director Early Independence Award (DP5-OD26386), and University of Pennsylvania NIH T32 Radiology Research Training Grant (5T32EB004311-12).
Objectives
There currently lacks a noninvasive and accurate method to distinguish benign and malignant ovarian lesion prior to treatment. This study developed a deep learning algorithm that ...distinguishes benign from malignant ovarian lesion by applying a convolutional neural network on routine MR imaging.
Methods
Five hundred forty-five lesions (379 benign and 166 malignant) from 451 patients from a single institution were divided into training, validation, and testing set in a 7:2:1 ratio. Model performance was compared with four junior and three senior radiologists on the test set.
Results
Compared with junior radiologists averaged, the final ensemble model combining MR imaging and clinical variables had a higher test accuracy (0.87 vs 0.64,
p
< 0.001) and specificity (0.92 vs 0.64,
p
< 0.001) with comparable sensitivity (0.75 vs 0.63,
p
= 0.407). Against the senior radiologists averaged, the final ensemble model also had a higher test accuracy (0.87 vs 0.74,
p
= 0.033) and specificity (0.92 vs 0.70,
p
< 0.001) with comparable sensitivity (0.75 vs 0.83,
p
= 0.557). Assisted by the model’s probabilities, the junior radiologists achieved a higher average test accuracy (0.77 vs 0.64, Δ = 0.13,
p
< 0.001) and specificity (0.81 vs 0.64, Δ = 0.17,
p
< 0.001) with unchanged sensitivity (0.69 vs 0.63, Δ = 0.06,
p
= 0.302). With the AI probabilities, the junior radiologists had higher specificity (0.81 vs 0.70, Δ = 0.11,
p
= 0.005) but similar accuracy (0.77 vs 0.74, Δ = 0.03,
p
= 0.409) and sensitivity (0.69 vs 0.83, Δ = -0.146,
p
= 0.097) when compared with the senior radiologists.
Conclusions
These results demonstrate that artificial intelligence based on deep learning can assist radiologists in assessing the nature of ovarian lesions and improve their performance.
Key Points
• Artificial Intelligence based on deep learning can assess the nature of ovarian lesions on routine MRI with higher accuracy and specificity than radiologists.
• Assisted by the deep learning model’s probabilities, junior radiologists achieved better performance that matched those of senior radiologists.
Thymic Foxp3-expressing regulatory T cells are activated by peripheral self-antigen to increase their suppressive function, and a fraction of these cells survive as memory regulatory T cells ...(mTregs). mTregs persist in nonlymphoid tissue after cessation of Ag expression and have enhanced capacity to suppress tissue-specific autoimmunity. In this study, we show that murine mTregs express specific effector memory T cell markers and localize preferentially to hair follicles in skin. Memory Tregs express high levels of both IL-2Rα and IL-7Rα. Using a genetic-deletion approach, we show that IL-2 is required to generate mTregs from naive CD4(+) T cell precursors in vivo. However, IL-2 is not required to maintain these cells in the skin and skin-draining lymph nodes. Conversely, IL-7 is essential for maintaining mTregs in skin in the steady state. These results elucidate the fundamental biology of mTregs and show that IL-7 plays an important role in their survival in skin.
Despite the success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies, successful targeting of solid tumors with CAR T cells has been limited by a lack of durable ...responses and reports of toxicities. Our understanding of the limited therapeutic efficacy in solid tumors could be improved with quantitative tools that allow characterization of CAR T-targeted antigens in tumors and accurate monitoring of response.
We used a radiolabeled FAP inhibitor (FAPI) 18FAlF-FAPI-74 probe to complement ongoing efforts to develop and optimize FAP CAR T cells. The selectivity of the radiotracer for FAP was characterized in vitro, and its ability to monitor changes in FAP expression was evaluated using rodent models of lung cancer.
18FAlF-FAPI-74 showed selective retention in FAP+ cells in vitro, with effective blocking of the uptake in presence of unlabeled FAPI. In vivo, 18FAlF-FAPI-74 was able to detect FAP expression on tumor cells as well as FAP+ stromal cells in the tumor microenvironment with a high target-to-background ratio. We further demonstrated the utility of the tracer to monitor changes in FAP expression following FAP CAR T-cell therapy, and the PET imaging findings showed a robust correlation with ex vivo analyses.
This noninvasive imaging approach to interrogate the tumor microenvironment represents an innovative pairing of a diagnostic PET probe with solid tumor CAR T-cell therapy and has the potential to serve as a predictive and pharmacodynamic response biomarker for FAP as well as other stroma-targeted therapies. A PET imaging approach targeting FAP expressed on activated fibroblasts of the tumor stroma has the potential to predict and monitor therapeutic response to FAP-targeted CAR T-cell therapy. See related commentary by Weber et al., p. 5241.
We have designed, synthesized, and characterized a library of 38 novel flavonoid compounds linked with amines. Some of these amine-linked flavonoids have potent
activity against parasites that cause ...cutaneous leishmaniasis, a tropical disease endemic in 80 countries worldwide. The most promising candidate,
, was highly active with IC
of 0.3 μM against
,
and
amastigotes. It was metabolically stable (39% and 66% of
remaining after 30-minute incubation with human and rat liver microsomes respectively). In
LV78 cutaneous leishmaniasis mouse model, intralesional injection of
(10 mg/kg, once every 4 days for 8 times) demonstrated promising effect in reducing the footpad lesion thickness by 72%, displaying an efficacy comparable to SSG (63%).
Chimeric antigen receptor (CAR) T cell therapy has been successful for hematological malignancies. Still, a lack of efficacy and potential toxicities have slowed its application for other ...indications. Furthermore, CAR T cells undergo dynamic expansion and contraction in vivo that cannot be easily predicted or controlled. Therefore, the safety and utility of such therapies could be enhanced by engineered mechanisms that engender reversible control and quantitative monitoring. Here, we use a genetic tag based on the enzyme Escherichia coli dihydrofolate reductase (eDHFR), and derivatives of trimethoprim (TMP) to modulate and monitor CAR expression and T cell activity. We fused eDHFR to the CAR C terminus, allowing regulation with TMP-based proteolysis-targeting chimeric small molecules (PROTACs). Fusion of eDHFR to the CAR does not interfere with cell signaling or its cytotoxic function, and the addition of TMP-based PROTACs results in a reversible and dose-dependent inhibition of CAR activity via the proteosome. We show the regulation of CAR expression in vivo and demonstrate imaging of the cells with TMP radiotracers. In vitro immunogenicity assays using primary human immune cells and overlapping peptide fragments of eDHFR showed no memory immune repertoire for eDHFR. Overall, this translationally-orientied approach allows for temporal monitoring and image-guided control of cell-based therapies.
Certain BH3-only proteins transiently bind and activate Bak and Bax, initiating their oligomerization and the permeabilization of the mitochondrial outer membrane, a pivotal step in the mitochondrial ...pathway to apoptosis. Here we describe the first crystal structures of an activator BH3 peptide bound to Bak and illustrate their use in the design of BH3 derivatives capable of inhibiting human Bak on mitochondria. These BH3 derivatives compete for the activation site at the canonical groove, are the first engineered inhibitors of Bak activation, and support the role of key conformational transitions associated with Bak activation.
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•First crystal structure of Bak bound to a BH3-only peptide•First inhibitor of Bak with a clearly defined molecular mechanism•BH3 binding is sufficient to disrupt Bak from the complex containing VDAC2•Ligands that form a stable complex with Bak can block its activation
Brouwer et al. describe the first crystal structures of BH3-only peptides bound to Bak, which reveal a cavity that may be important for activation. They then use structure-based design to alter the Bim-BH3 peptide and convert it from an activator to an inhibitor of Bak.
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