BACKGROUND. Neoantigen-driven recognition and T cell-mediated killing contribute to tumor clearance following adoptive cell therapy (ACT) with Tumor-Infiltrating Lymphocytes (TILs). Yet, how diversity, frequency, and persistence of expanded neoepitope-specific CD8+ T cells derived from TIL infusion products affect patient outcome is not fully determined. METHODS. Using barcoded pMHC multimers, we provide a comprehensive mapping of CD8+ T cells recognizing neoepitopes in TIL infusion products and blood samples from 26 metastatic mela-noma patients who received ACT. RESULTS. We identified 106 neoepitopes within TIL infusion products corresponding to 1.8% of all predicted neoepitopes. We observed neoepitope-specific recognition to be virtually devoid in TIL infusion products given to patients with progressive disease outcome. Moreover, we found that the frequency of neoepitope-specific CD8+ T cells in TIL infusion products correlated with in-creased survival, and that detection of engrafted CD8+ T cells in post-treatment (i.e. originating from the TIL infusion product) were unique to responders of TIL-ACT. Finally, we found that a transcriptional signature for lymphocyte activity within the tumor microenvironment was associated with a higher frequency of neoepitope-specific CD8+ T cells in the infusion product. CONCLUSIONS. These data support previous case studies of neoepitope-specific CD8+ T cells in melanoma, and indicate that successful TIL-ACT is associated with an expansion of neoepitope-specific CD8+ T cells. FUNDING. NEYE Foundation; European Research Council; Lundbeck Foundation Fellowship; Carlsberg Foundation.
Nikolaj Pagh Kristensen, Christina Heeke, Siri A. Tvingsholm, Annie Borch, Arianna Draghi, Michael D. Crowther, Ibel Carri, Kamilla K. Munk, Jeppe Sejerø Holm, Anne-Mette Bjerregaard, Amalie Kai Bentzen, Andrea M. Marquard, Zoltan Szallasi, Nicholas McGranahan, Rikke Andersen, Morten Nielsen, Göran B. Jönsson, Marco Donia, Inge Marie Svane, Sine Reker Hadrup
The tumorigenic mechanism for pancreatic ductal adenocarcinoma (PDAC) is not clear, although chronic inflammation is implicated. Here, we identified an inflammatory cytokine–regulated transfer RNA–derived (tRNA-derived) fragment, tRF-21-VBY9PYKHD (tRF-21), as a tumor suppressor in PDAC progression. We found that the biogenesis of tRF-21 could be inhibited by leukemia inhibitory factor and IL-6 via the splicing factor SRSF5. Reduced tRF-21 promoted AKT2/1-mediated heterogeneous nuclear ribonucleoprotein L (hnRNP L) phosphorylation, enhancing hnRNP L to interact with dead-box helicase 17 (DDX17) to form an alternative splicing complex. The provoked hnRNP L-DDX17 activity preferentially spliced Caspase 9 and mH2A1 pre-mRNAs to form Caspase 9b and mH2A1.2, promoting PDAC cell malignant phenotypes. The tRF-21 levels were significantly lower in PDACs than in normal tissues, and patients with low tRF-21 levels had a poor prognosis. Treatment of mouse PDAC xenografts or patient-derived xenografts (PDXs) with tRF-21 mimics repressed tumor growth and metastasis. These results demonstrate that tRF-21 has a tumor-suppressive effect and is a potential therapeutic agent for PDAC.
Ling Pan, Xudong Huang, Ze-Xian Liu, Ying Ye, Rui Li, Jialiang Zhang, Guandi Wu, Ruihong Bai, Lisha Zhuang, Lusheng Wei, Mei Li, Yanfen Zheng, Jiachun Su, Junge Deng, Shuang Deng, Lingxing Zeng, Shaoping Zhang, Chen Wu, Xu Che, Chengfeng Wang, Rufu Chen, Dongxin Lin, Jian Zheng
Contrasting with the predicted anorexigenic effect of increasing brain serotonin signaling, long-term use of selective serotonin reuptake inhibitors (SSRIs) antidepressants correlates with body weight gain. This adverse outcome increases the risk of transitioning to obesity and interferes with treatment compliance. Here we show that orally administered fluoxetine (Flx), a widely prescribed SSRI, increased body weight by enhancing food intake in healthy mice at two different time points and through two distinct mechanisms. Within hours, Flx decreased the activity of a subset of brainstem serotonergic neurons by triggering autoinhibitory signaling through the Htr1a receptor. Upon longer treatment Flx blunted Htr2c expression/signaling, decreased the phosphorylation of Creb and Stat3 and dampened the production of POMC/α-MSH in hypothalamic neurons, thereby increasing food intake. Accordingly, exogenous stimulation of the melanocortin 4 receptor (MC4R) by co-treating mice with Flx and lipocalin-2, an anorexigenic hormone signaling through this receptor, normalized feeding and body weight. Flx and other SSRIs also inhibit CREB/STAT3 phosphorylation in a human neuronal cell line suggesting that these non-canonical effects could also occur in long-term users of SSRIs. By defining the molecular basis of the long-term SSRIs-associated weight gain this study proposes a therapeutic strategy to counter it.
Maria Jose Ortuno, Marc Schneeberger, Anoj Ilanges, François Marchildon, Kyle Pellegrino, Jeffrey M. Friedman, Patricia Ducy
Human monoclonal antibodies were used here to study the mechanism of neuron intoxication by tetanus neurotoxin and to evaluate them as a safe preventive and therapeutic substitute of hyperimmune sera for tetanus in mice. By screening memory B cells of immune donors, we selected two monoclonal antibodies specific for tetanus neurotoxin with exceptionally high neutralizing activities, which were extensively characterized both structurally and functionally. We found that these antibodies interfere with the binding and translocation of the neurotoxin into neurons by interacting with two epitopes, whose definition pinpoints crucial events in the cellular pathogenesis of tetanus. This information explains the unprecedented neutralization ability of these antibodies, which were found to be exceptionally potent in preventing experimental tetanus when injected in mice long before the neurotoxin. Moreover, their Fab derivatives neutralized tetanus neurotoxin in post-exposure experiments, suggesting their potential therapeutic use via intrathecal injection. As such, these human monoclonal antibodies, as well as their Fab derivatives, meet all requirements for being considered for prophylaxis and therapy of human tetanus and are ready for clinical trials.
Marco Pirazzini, Alessandro Grinzato, Davide Corti, Sonia Barbieri, Oneda Leka, Francesca Vallese, Marika Tonellato, Chiara Silacci-Fregni, Luca Piccoli, Eaazhisai Kandiah, Giampietro Schiavo, Giuseppe Zanotti, Antonio Lanzavecchia, Cesare Montecucco
NKTR-255 is a novel polyethylene glycol (PEG)-conjugate of recombinant human IL-15 (rhIL-15) being examined as a potential cancer immunotherapeutic. Since IL-15 responses can be mediated by trans- or cis-presentation via IL-15Rα or soluble IL-15/IL-15Rα complexes, we investigated the role of IL-15Rα in driving NKTR-255 responses using defined naïve and memory ovalbumin-specific CD8 T cells (OT-I) CD8 T and NK cells in mice. NKTR-255 induced a 2.5 and 2.0-fold expansion of CD8 T and NK cells, respectively in WT mice. In adoptive transfer studies, proliferation of naïve and memory Wt OT-I T cells in response to NKTR-255 was not impaired in IL-15Rα−/− mice, suggesting trans-presentation was not utilized by NKTR-255. Interestingly, naïve IL-15Rα−/− OT-I cells had deficient responses to NKTR-255 while memory IL-15Rα−/− OT-I cell responses were partially impaired, suggesting that naive CD8 T cells are more dependent on cis-presentation of NKTR-255 than memory CD8 T cells. In bone marrow chimeras studies, IL-15Rα−/− and WT NK cells present in WT recipients had similar responses to NKTR-255, suggesting that cis-presentation is not utilized by NK cells. NKTR-255 could form soluble complexes with IL-15Rα; binding to murine IL-15Rα generated superagonists that preferentially stimulated NK cells showing that conversion to IL-15Rβ agonist biases the response towards NK cells. These findings highlight the ability of NKTR-255 to utilize IL-15Rα for cis-presentation and act as an IL-15Rαβ agonist on CD8 T cells.
Tanya O. Robinson, Shweta M. Hegde, Allison J. Chang, Achintyan Gangadharan, Sarai Rivas, Loui Madakamutil, Jonathan Zalevsky, Takahiro Miyazaki, Kimberly S. Schluns
Alcohol use disorder (AUD) is associated with substantial morbidity, mortality, and societal cost, and pharmacological treatment options for AUD are limited. The endogenous cannabinoid (eCB) signaling system is critically involved in reward processing and alcohol intake is positively correlated with release of the eCB ligand 2-Arachidonoylglycerol (2-AG) within reward neurocircuitry. Here we show that genetic and pharmacological inhibition of diacylglycerol lipase (DAGL), the rate limiting enzyme in the synthesis of 2-AG, reduces alcohol consumption in a variety of preclinical models ranging from a voluntary free-access model to aversion resistant-drinking and dependence-like drinking induced via chronic intermittent ethanol vapor exposure in mice. DAGL inhibition during either chronic alcohol consumption or protracted withdrawal was devoid of anxiogenic and depressive-like behavioral effects. Lastly, DAGL inhibition also prevented ethanol-induced suppression of GABAergic transmission onto midbrain dopamine neurons, providing mechanistic insight into how DAGL inhibition could affect alcohol reward. These data suggest reducing 2-AG signaling via inhibition of DAGL could represent an effective approach to reduce alcohol consumption across the spectrum of AUD severity.
Nathan D. Winters, Gaurav Bedse, Anastasia A. Astafyev, Toni A. Patrick, Megan Altemus, Amanda J. Morgan, Snigdha Mukerjee, Keenan D. Johnson, Vikrant R. Mahajan, Md. Jashim Uddin, Philip J. Kingsley, Samuel W. Centanni, Cody A. Siciliano, David C. Samuels, Lawrence J. Marnett, Danny G. Winder, Sachin Patel
Androgen receptor (AR)-positive prostate cancers (PCa) and estrogen receptor (ER)-positive luminal breast cancers (BCa) are generally less responsive to immunotherapy compared to certain tumor types such as melanoma. However, the underlying mechanisms are not fully elucidated. Here we found that FOXA1 overexpression inversely correlated with interferon (IFN) signature and antigen presentation gene expression in PCa and BCa patients. FOXA1 bound STAT2 DNA binding domain and suppressed STAT2 DNA binding activity, IFN signaling gene expression and cancer immune response independently of the transactivation activity of FOXA1 and its mutations detected in prostate and breast cancers. Increased FOXA1 expression promoted cancer immuno- and chemotherapy resistance in mice and PCa and BCa patients. These findings were also validated in bladder cancer expressing high level FOXA1. FOXA1 overexpression could be a prognostic factor to predict therapy resistance and a viable target to sensitize luminal prostate, breast and bladder cancer to immuno- and chemotherapy.
Yundong He, Liguo Wang, Ting Wei, Yu-Tian Xiao, Haoyue Sheng, Hengchuan Su, Daniel P. Hollern, Xiaoling Zhang, Jian Ma, Simeng Wen, Hongyan Xie, Yuqian Yan, Yunqian Pan, Xiaonan Hou, Xiaojia Tang, Vera J. Suman, Jodi M. Carter, Richard Weinshilboum, Liewei Wang, Krishna R. Kalari, Saravut J. Weroha, Alan H. Bryce, Judy C. Boughey, Haidong Dong, Charles M. Perou, Dingwei Ye, Matthew P. Goetz, Shancheng Ren, Haojie Huang
Tirzepatide (LY3298176), a dual GIP and GLP-1 receptor agonist, delivered superior glycemic control and weight loss compared to GLP-1 receptor (GLP-1R) agonism in patients with type 2 diabetes. However, the mechanism by which tirzepatide improves efficacy and how GIP receptor (GIPR) agonism contributes is not fully understood. Here, we show that tirzepatide is an effective insulin sensitizer, improving insulin sensitivity in obese mice to a greater extent than GLP-1R agonism. To determine if GIPR agonism contributes, we compared the effect of tirzepatide in obese wild-type and Glp-1r null mice. In the absence of GLP-1R-induced weight loss, tirzepatide improved insulin sensitivity by enhancing glucose disposal in white adipose tissue (WAT). In support, a long-acting GIPR agonist (LAGIPRA) was found to enhance insulin sensitivity by augmenting glucose disposal in WAT. Interestingly, the effect of tirzepatide and LAGIPRA on insulin sensitivity was associated with reduced branched-chain amino (BCAAs) and keto-acids in the circulation. Insulin sensitization was associated with upregulation of genes associated with the catabolism of glucose, lipid and BCAAs in brown adipose tissue. Together, our studies show that tirzepatide improved insulin sensitivity in a weight-dependent and -independent manner. These results highlight how GIPR agonism contributes to the therapeutic profile of dual receptor agonism, offering mechanistic insights into the clinical efficacy of tirzepatide.
Ricardo J. Samms, Michael E. Christe, Kyla A. Collins, Valentina Pirro, Brian A. Droz, Adrienne K. Holland, Jessica L. Friedrich, Samantha Wojnicki, Debra L. Konkol, Richard Cosgrove, Ellen P.S. Conceição Furber, Xiaoping Ruan, Libbey S. O'Farrell, Annie M. Long, Mridula Dogra, Jill A. Willency, Yanzhu Lin, Liyun Ding, Christine C. Cheng, Over Cabrera, Daniel A. Briere, Jorge Alsina-Fernandez, Ruth E. Gimeno, Julie S. Moyers, Tamer Coskun, Matthew P. Coghlan, Kyle W. Sloop, William C. Roell
Adoptive T cell therapies (ACTs) hold great promise in cancer treatment, but low overall response rates in patients with solid tumors underscore remaining challenges in realizing the potential of this cellular immunotherapy approach. Promoting CD8+ T cell adaptation to tissue residency represents an underutilized but promising strategy to improve tumor-infiltrating lymphocyte (TIL) function. Here, we report that deletion of the HIF negative regulator von Hippel-Lindau (VHL) in CD8+ T cells induced HIF-1α/HIF-2α–dependent differentiation of tissue-resident memory–like (Trm-like) TILs in mouse models of malignancy. VHL-deficient TILs accumulated in tumors and exhibited a core Trm signature despite an exhaustion-associated phenotype, which led to retained polyfunctionality and response to αPD-1 immunotherapy, resulting in tumor eradication and protective tissue-resident memory. VHL deficiency similarly facilitated enhanced accumulation of chimeric antigen receptor (CAR) T cells with a Trm-like phenotype in tumors. Thus, HIF activity in CD8+ TILs promotes accumulation and antitumor activity, providing a new strategy to enhance the efficacy of ACTs.
Ilkka Liikanen, Colette Lauhan, Sara Quon, Kyla Omilusik, Anthony T. Phan, Laura Barceló Bartrolí, Amir Ferry, John Goulding, Joyce Chen, James P. Scott-Browne, Jason T. Yustein, Nicole E. Scharping, Deborah A. Witherden, Ananda W. Goldrath
The A3 adenosine receptor (A3AR) has emerged as a therapeutic target with A3AR agonists to tackle the global challenge of neuropathic pain; investigation into their mode of action is essential for ongoing clinical development. A3ARs on immune cells, and their activation during pathology, modulates cytokine release. Thus, immune cells as a cellular substrate for the pharmacological action of A3AR agonists is enticing but unknown. Studies herein discovered that RagKO mice lacking T- and B-cells are insensitive to the anti-allodynic effects of A3AR agonists versus wild-type (WT) mice. Similar findings were observed in interleukin-10 and interleukin-10 receptor knockout mice. Adoptive transfer of CD4+ T-cells (CD4+-T) from WT mice infiltrated the dorsal root ganglion (DRG) and restored A3AR agonist-mediated anti-allodynia in RagKO mice; CD4+-T from Adora3KO or Il10KO mice did not. Transfer of CD4+-T from WT, but not Il10KO, into Il10KO mice fully reinstated anti-allodynic effects of A3AR activation. Transfer of CD4+-T from WT, but not Il10KO, into Adora3KO mice fully reinstated anti-allodynic effects of A3AR activation. Notably, A3AR agonism reduced DRG neuron excitability when co-cultured with CD4+-T in an IL-10-dependent manner. A3AR actions on CD4+-T infiltrate in the DRG decreased phosphorylation of GluN2B-containing N‐methyl‐D‐aspartate receptors at Tyr1472, a modification associated with regulating neuronal hypersensitivity. Our findings establish that activation of A3AR on CD4+-T cells to release of IL-10 is required and sufficient for A3AR agonists as therapeutics.
Mariaconcetta Durante, Silvia Squillace, Filomena Lauro, Luigino Antonio Giancotti, Elisabetta Coppi, Federica Cherchi, Lorenzo Di Cesare Mannelli, Carla Ghelardini, Grant Kolar, Carrie Wahlman, Adeleye Opejin, Cuiying Xiao, Marc L. Reitman, Dilip K. Tosh, Daniel Hawiger, Kenneth A. Jacobson, Daniela Salvemini
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