We investigated human T-cell repertoire formation using high throughput TCRβ CDR3 sequencing in immunodeficient mice receiving human hematopoietic stem cells (HSCs) and human thymus grafts. Replicate humanized mice generated diverse and highly divergent repertoires. Repertoire narrowing and increased CDR3β sharing was observed during thymocyte selection. While hydrophobicity analysis implicated self-peptides in positive selection of the overall repertoire, positive selection favored shorter shared sequences that had reduced hydrophobicity at positions 6 and 7 of CDR3βs, suggesting weaker interactions with self-peptides than unshared sequences, possibly allowing escape from negative selection. Sharing was similar between autologous and allogeneic thymi and occurred between different cell subsets. Shared sequences were enriched for allo-crossreactive CDR3βs and for Type 1 diabetes-associated autoreactive CDR3βs. Single-cell TCR-sequencing showed increased sharing of CDR3αs compared to CDR3βs between mice. Our data collectively implicate preferential positive selection for shared human CDR3βs that are highly cross-reactive. While previous studies suggested a role for recombination bias in producing “public” sequences in mice, our study is the first to demonstrate a role for thymic selection. Our results implicate positive selection for promiscuous TCRβ sequences that likely evade negative selection, due to their low affinity for self-ligands, in the abundance of “public” human TCRβ sequences.
Mohsen Khosravi-Maharlooei, Aleksandar Obradovic, Aditya Misra, Keshav Motwani, Markus Holzl, Howard R. Seay, Susan DeWolf, Grace Nauman, Nichole Danzl, Haowei Li, Siu-hong Ho, Robert Winchester, Yufeng Shen, Todd M. Brusko, Megan Sykes
Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their prevalence in various diseases including cancer. Drug development targeting IDPs is challenging because they have dynamical structure features and conventional drug design is not applicable. NUPR1 is an IDP playing an important role in pancreatic cancer. We previously reported that Trifluoperazine (TFP), an antipsychotic agent, was capable of binding to NUPR1 and inhibiting tumors growth. Unfortunately, TFP showed strong central nervous system side-effects. In this work, we undertook a multidisciplinary approach to optimize TFP, based on the synergy of computer modeling, chemical synthesis, and a variety of biophysical, biochemical and biological evaluations. A family of TFP-derived compounds was produced and the most active one, named ZZW-115, showed a dose-dependent tumor regression with no neurological effects and induced cell death mainly by necroptosis. This study opens a new perspective for drug development against IDPs, demonstrating the possibility of successful ligand-based drug design for such challenging targets.
Patricia Santofimia-Castaño, Yi Xia, Wenjun Lan, Zhengwei Zhou, Can Huang, Ling Peng, Philippe Soubeyran, Adrian Velazquez-Campoy, Olga Abian, Bruno Rizzuti, Jose L. Neira, Juan Iovanna
BACKGROUND. African American (AA) patients have higher cancer mortality rates and shorter survival times compared to European American (EA) patients. Despite a significant focus on socioeconomic factors, recent findings strongly argue the existence of biological factors driving this disparity. Most of these factors have been described in a cancer-type specific context rather than a pan-cancer setting. METHODS. A novel in silico approach based on Gene Set Enrichment Analysis (GSEA) coupled to Transcription Factor enrichment was carried out to identify common biological drivers of pan-cancer racial disparity using The Cancer Genome Atlas (TCGA) dataset. Mitochondrial content in patient tissues was examined using a multi-cancer tissue microarray approach (TMA). RESULTS. Mitochondrial oxidative phosphorylation was uniquely enriched in AA tumors compared to EA tumors across various cancer types. AA tumors also showed strong enrichment for the ERR1-PGC1α-mediated transcriptional program, which has been implicated in mitochondrial biogenesis. TMA analysis revealed that AA cancers harbor significantly more mitochondria compared to their EA counterparts. CONCLUSIONS. These findings highlight changes in mitochondria as a common distinguishing feature between AA and EA tumors in a pan-cancer setting, and provide the rationale for the repurposing of mitochondrial inhibitors to treat AA cancers.
Danthasinghe Waduge Badrajee Piyarathna, Akhila Balasubramanian, James M. Arnold, Stacy M. Lloyd, Balasubramanyam Karanam, Patricia Castro, Michael M. Ittmann, Nagireddy Putluri, Nora Navone, Jeffrey A. Jones, Wendong Yu, Vlad C. Sandulache, Andrew G. Sikora, George Michailidis, Arun Sreekumar
Acute kidney injury (AKI) can lead to chronic kidney disease (CKD) if injury is severe and/or repair is incomplete. However, the pathogenesis of CKD following renal ischemic injury is not fully understood. Capillary rarefaction and tubular hypoxia are common findings during the AKI to CKD transition. We investigated the tubular stress response to hypoxia and demonstrated that a stress responsive transcription factor, FoxO3, was regulated by prolyl hydroxylase. Hypoxia inhibited FoxO3 prolyl hydroxylation and FoxO3 degradation, thus leading to FoxO3 accumulation and activation in tubular cells. Hypoxia-activated Hif-1α contributed to FoxO3 activation and functioned to protect kidneys, as tubular deletion of Hif-1α decreased hypoxia-induced FoxO3 activation, and resulted in more severe tubular injury and interstitial fibrosis following ischemic injury. Strikingly, tubular deletion of FoxO3 during the AKI to CKD transition aggravated renal structural and functional damage leading to a more profound CKD phenotype. We showed that tubular deletion of FoxO3 resulted in decreased autophagic response and increased oxidative injury, which may explain renal protection by FoxO3. Our study indicates that in the hypoxic kidney, stress responsive transcription factors can be activated for adaptions to counteract hypoxic insults, thus attenuating CKD development.
Ling Li, Huimin Kang, Qing Zhang, Vivette D. D'Agati, Qais Al-Awqati, Fangming Lin
Rationale Tumor infiltrating lymphocytes are widely associated with positive outcomes, yet carry key indicators of a systemic failed immune response against unresolved cancer. Cancer immunotherapies can reverse their tolerance phenotypes, while preserving tumor-reactivity and neoantigen-specificity shared with circulating immune cells. Objectives We performed comprehensive transcriptomic analyses to identify gene signatures common to circulating and tumor infiltrating lymphocytes in the context of clear cell renal cell carcinoma. Modulated genes also associated with disease outcome were validated in other cancer types. Findings Using bioinformatics, we identified practical diagnostic markers and actionable targets of the failed immune response. On circulating lymphocytes, three genes, LEF1, FASLG, and MMP9, could efficiently stratify patients from healthy control donors. From their associations with resistance to cancer immunotherapies and microbial infections, we uncovered not only pan-cancer, but pan-pathology failed immune response profiles. A prominent lymphocytic matrix metallopeptidase cell migration pathway, is central to a panoply of diseases and tumor immunogenicity, correlates with multi-cancer recurrence, and identifies a feasible, non-invasive approach to pan-pathology diagnoses. Conclusions The non-invasive differently expressed genes we have identified warrant future investigation towards the development of their potential in precision diagnostics and precision pan-disease immunotherapeutics.
Anne Monette, Antigoni Morou, Nadia A. Al-Banna, Louise Rousseau, Jean-Baptiste Lattouf, Sara Rahmati, Tomas Tokar, Jean-Pierre Routy, Jean-Francois Cailhier, Daniel E. Kaufmann, Igor Jurisica, Rejean Lapointe
Identifying non-addictive opioid medications is a high priority in medical sciences, but μ-opioid receptors mediate both the analgesic and addictive effects of opioids. We found a significant pharmacodynamic difference between morphine and methadone that is determined entirely by heteromerization of μ-opioid receptors with galanin Gal1 receptors, rendering a profound decrease in the potency of methadone. This was explained by methadone’s weaker proficiency to activate the dopaminergic system as compared to morphine and predicted a dissociation of therapeutic versus euphoric effects of methadone, which was corroborated by a significantly lower incidence of self-report of “high” in methadone-maintained patients. These results suggest that μ-opioid-Gal1 receptor heteromers mediate the dopaminergic effects of opioids that may lead to a lower addictive liability of opioids with selective low potency for the μ-opioid-Gal1 receptor heteromer, exemplified by methadone.
Ning-Sheng Cai, César Quiroz, Jordi Bonaventura, Alessandro Bonifazi, Thomas O. Cole, Julia Purks, Amy S. Billing, Ebonie Massey, Michael Wagner, Eric D. Wish, Xavier Guitart, William Rea, Sherry Lam, Estefanía Moreno, Verònica Casadó-Anguera, Aaron D. Greenblatt, Arthur E. Jacobson, Kenner C. Rice, Vicent Casadó, Amy H. Newman, John W. Winkelman, Michael Michaelides, Eric Weintraub, Nora D. Volkow, Annabelle M. Belcher, Sergi Ferré
Post-transplantation cyclophosphamide (PTCy) recently has had a marked impact on human allogeneic hematopoietic cell transplantation (HCT). Yet, our understanding of how PTCy prevents graft-versus-host disease (GVHD) largely has been extrapolated from major histocompatibility complex (MHC)-matched murine skin allografting models that were highly contextual in their efficacy. Herein, we developed a T-cell-replete, MHC-haploidentical, murine HCT model (B6C3F1→B6D2F1) to test the putative underlying mechanisms: alloreactive T-cell elimination, alloreactive T-cell intrathymic clonal deletion, and suppressor T-cell induction. In this model and confirmed in four others, PTCy did not eliminate alloreactive T cells identified using either specific Vβs or the 2C or 4C T-cell receptors. Furthermore, the thymus was not necessary for PTCy’s efficacy. Rather, PTCy induced alloreactive T-cell functional impairment which was supported by highly active suppressive mechanisms established within one day after PTCy that were sufficient to prevent new donor T cells from causing GVHD. These suppressive mechanisms included the rapid, preferential recovery of CD4+CD25+Foxp3+ regulatory T cells, including those that were alloantigen-specific, which served an increasingly critical function over time. Our results prompt a paradigm-shift in our mechanistic understanding of PTCy. These results have direct clinical implications for understanding tolerance induction and for rationally developing novel strategies to improve patient outcomes.
Lucas P. Wachsmuth, Michael T. Patterson, Michael A. Eckhaus, David J. Venzon, Ronald E. Gress, Christopher G. Kanakry
Opioid use disorder (OUD) is associated with the emergence of persistent negative emotional states during drug abstinence that drive compulsive drug taking and seeking. Functional magnetic resonance imaging (fMRI) in rats identified neurocircuits that were activated by stimuli that were previously paired with heroin withdrawal. The activation of amygdala and hypothalamic circuits was related to the degree of heroin dependence, supporting the significance of conditioned negative affect in sustaining compulsive-like heroin seeking and taking and providing neurobiological insights into the drivers of the current opioid crisis.
Stephanie A. Carmack, Robin J. Keeley, Janaina C.M. Vendruscolo, Emily G. Lowery-Gionta, Hanbing Lu, George F. Koob, Elliot A. Stein, Leandro F. Vendruscolo
Background: Chimeric antigen receptor (CAR) T cells are a promising therapy for hematologic malignancies. B-cell maturation antigen (BCMA) is a rational target in multiple myeloma (MM). Methods: We conducted a phase I study of autologous T cells lentivirally-transduced with a fully-human, BCMA-specific CAR containing CD3ζ and 4-1BB signaling domains (CART-BCMA), in subjects with relapsed/refractory MM. Twenty-five subjects were treated in 3 cohorts: 1) 1-5 x 108 CART-BCMA cells alone; 2) Cyclophosphamide (Cy) 1.5 g/m2 + 1-5 x 107 CART-BCMA cells; and 3) Cy 1.5 g/m2 + 1-5 x 108 CART-BCMA cells. No pre-specified BCMA expression level was required. Results: CART-BCMA cells were manufactured and expanded in all subjects. Toxicities included cytokine release syndrome and neurotoxicity, which were grade 3-4 in 8 (32%) and 3 (12%) subjects, respectively, and reversible. One subject died at day 24 from candidemia and progressive myeloma, following treatment for severe CRS and encephalopathy. Responses (based on treated subjects) were seen in 4/9 (44%) in cohort 1, 1/5 (20%) in cohort 2, and 7/11 (64%) in cohort 3, including 5 partial, 5 very good partial, and 2 complete responses, 3 of which were ongoing at 11, 14, and 32 months. Decreased BCMA expression on residual MM cells was noted in responders; expression increased at progression in most. Responses and CART-BCMA expansion were associated with CD4:CD8 T cell ratio and frequency of CD45RO-CD27+CD8+ T cells in the pre-manufacturing leukapheresis product. Conclusion: CART-BCMA infusions with or without lymphodepleting chemotherapy are clinically active in heavily-pretreated MM patients. Trial Registration: NCT02546167. Funding: University of Pennsylvania-Novartis Alliance and NIH.
Adam D. Cohen, Alfred L. Garfall, Edward A. Stadtmauer, J. Joseph Melenhorst, Simon F. Lacey, Eric Lancaster, Dan T. Vogl, Brendan M. Weiss, Karen Dengel, Annemarie Nelson, Gabriela Plesa, Fang Chen, Megan M. Davis, Wei-Ting Hwang, Regina M. Young, Jennifer L. Brogdon, Randi Isaacs, Iulian Pruteanu-Malinici, Don L. Siegel, Bruce L. Levine, Carl H. June, Michael C. Milone
Mice selectively expressing PPARγ dominant negative mutation in vascular smooth muscle exhibit RhoBTB1-deficiency and hypertension. Our rationale was to employ genetic complementation to uncover the mechanism of action of RhoBTB1 in vascular smooth muscle. Inducible smooth muscle-specific restoration of RhoBTB1 fully corrected the hypertension and arterial stiffness by improving vasodilator function. Notably, the cardiovascular protection occurred despite preservation of increased agonist-mediated contraction and RhoA/Rho kinase activity, suggesting RhoBTB1 selectively controls vasodilation. RhoBTB1 augmented the cGMP response to nitric oxide by restraining the activity of phosphodiesterase 5 (PDE5) by acting as a substrate adaptor delivering PDE5 to the Cullin-3 E3 Ring ubiquitin ligase complex for ubiquitination inhibiting PDE5. Angiotensin-II infusion also caused RhoBTB1-deficiency and hypertension which was prevented by smooth muscle specific RhoBTB1 restoration. We conclude that RhoBTB1 protected from hypertension, vascular smooth muscle dysfunction, and arterial stiffness in at least two models of hypertension.
Masashi Mukohda, Shi Fang, Jing Wu, Larry N. Agbor, Anand R. Nair, Stella-Rita C. Ibeawuchi, Chunyan Hu, Xuebo Liu, Ko-Ting Lu, Deng-Fu Guo, Deborah R. Davis, Henry L. Keen, Frederick W. Quelle, Curt D. Sigmund
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