The efficacy of chimeric antigen receptor (CAR)-T therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28-EGFRvIII glioma revealed impaired mitochondrial ATP production and a markedly hypoxic status compared to ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of AMPK activator Metformin and the mTOR inhibitor Rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-gamma coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective anti-glioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28-EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group with fewer Ly6c+ CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.
Ryusuke Hatae, Keith Kyewalabye, Akane Yamamichi, Tiffany Chen, Su Phyu, Pavlina Chuntova, Takahide Nejo, Lauren S. Levine, Matthew H. Spitzer, Hideho Okada
Immune checkpoint inhibitors (ICIs) are indicated for a diverse range of cancer types, and characterizing the tumor immune microenvironment is critical for optimizing therapeutic strategies, including ICIs. T cell infiltration and activation status in the tumor microenvironment greatly affects the efficacy of ICIs. Here, we show that semaphorin 6D (Sema6D) forward signaling, which is reportedly involved in coordinating the orientation of cell development and migration as a guidance factor, impaired the infiltration and activation of tumor-specific CD8+ T cells in murine oral tumors. Sema6D expressed by nonhematopoietic cells was responsible for this phenotype. Plexin-A4, a receptor for Sema6D, inhibited T cell infiltration and partially suppressed CD8+ T cell activation and proliferation induced by Sema6D stimulation. Moreover, mouse oral tumors, which are resistant to PD-1–blocking treatment in wild-type mice, showed a response to the treatment in Sema6d-KO mice. Finally, analyses of public data sets of human head and neck squamous cell carcinoma, pan-cancer cohorts, and a retrospective cohort study showed that SEMA6D was mainly expressed by nonhematopoietic cells such as cancer cells, and SEMA6D expression was significantly negatively correlated with CD8A, PDCD1, IFNG, and GZMB expression. Thus, targeting Sema6D forward signaling is a promising option for increasing ICI efficacy.
Takashi Hirai, Yujiro Naito, Shohei Koyama, Yoshimitsu Nakanishi, Kentaro Masuhiro, Mayuko Izumi, Tomoki Kuge, Maiko Naito, Yumiko Mizuno, Yuta Yamaguchi, Sujin Kang, Moto Yaga, Yu Futami, Satoshi Nojima, Masayuki Nishide, Takayoshi Morita, Yasuhiro Kato, Takeshi Tsuda, Norihiko Takemoto, Yumi Kinugasa-Katayama, Taiki Aoshi, Jordan Kelly Villa, Kazuo Yamashita, Tomohiro Enokida, Yuta Hoshi, Kazuto Matsuura, Makoto Tahara, Hyota Takamatsu, Yoshito Takeda, Hidenori Inohara, Atsushi Kumanogoh
While the function of many leukocytes in transplant biology has been well defined, the role of eosinophils is controversial and remains poorly explored. Conflicting data exist regarding eosinophils’ role in alloimmunity. Due to their prevalence in the lung, and their defined role in other pulmonary pathologies such as asthma, we set out to explore the role of eosinophils in the long-term maintenance of the lung allograft. We noted that depletion of eosinophils results in the generation of donor-specific antibodies. Eosinophil depletion increased memory B cell, plasma cell, and antibody-secreting cell differentiation and resulted in de novo generation of follicular germinal centers. Germinal center formation depended on the expansion of CD4+Foxp3–Bcl6+CXCR5+PD-1+ T follicular helper (Tfh) cells, which increase in number after eosinophil depletion. Mechanistically, we demonstrate that eosinophils prevent Tfh cell generation by acting as the dominant source of IFN-γ in an established lung allograft, thus facilitating Th1 rather than Tfh polarization of naive CD4+ T cells. Our data thus describe what we believe is a unique and previously unknown role for eosinophils in maintaining allograft tolerance and suggest that indiscriminate administration of eosinophil-lytic corticosteroids for treatment of acute cellular rejection may inadvertently promote humoral alloimmunity.
Zhongcheng Mei, May A. Khalil, Yizhan Guo, Dongge Li, Anirban Banerjee, Yuriko Terada, Yuhei Yokoyama, Christina Kratzmeier, Kelly Chen, Lushen Li, Christine L. Lau, Jean-Paul Courneya, Irina G. Luzina, Sergei P. Atamas, Andrew E. Gelman, Daniel Kreisel, Elizabeth A. Jacobsen, Alexander S. Krupnick
The role of long noncoding RNAs (lncRNAs) in disease is incompletely understood, but their regulation of inflammation is increasingly appreciated. We addressed the extent of lncRNA involvement in inflammatory bowel disease (IBD) using biopsy-derived RNA-sequencing data from a large cohort of deeply phenotyped patients with IBD. Weighted gene correlation network analysis revealed gene modules of lncRNAs coexpressed with protein-coding genes enriched for biological pathways, correlated with epithelial and immune cell signatures, or correlated with distal colon expression. Correlation of modules with clinical features uncovered a module correlated with disease severity, with an enriched interferon response signature containing the hub lncRNA IRF1-AS1. Connecting genes to IBD-associated single nucleotide polymorphisms (SNPs) revealed an enrichment of SNP-adjacent lncRNAs in biologically relevant modules. Ulcerative colitis–specific SNPs were enriched in distal colon–related modules, suggesting that disease-specific mechanisms may result from altered lncRNA expression. The function of the IBD-associated SNP-adjacent lncRNA IRF1-AS1 was explored in human myeloid cells, and our results suggested IRF1-AS1 promoted optimal production of TNF-α, IL-6, and IL-23. A CRISPR/Cas9-mediated activation screen in THP-1 cells revealed several lncRNAs that modulated LPS-induced TNF-α responses. Overall, this study uncovered the expression patterns of lncRNAs in IBD that identify functional, disease-relevant lncRNAs.
John L. Johnson, Davit Sargsyan, Eric M. Neiman, Amy Hart, Aleksandar Stojmirovic, Roman Kosoy, Haritz Irizar, Mayte Suárez-Fariñas, Won-Min Song, Carmen Argmann, Stefan Avey, Liraz Shmuel-Galia, Tim Vierbuchen, Gerold Bongers, Yu Sun, Leonard Edelstein, Jacqueline Perrigoue, Jennifer E. Towne, Aisling O’Hara Hall, Katherine A. Fitzgerald, Kasper Hoebe
Although cold preservation remains the gold standard in organ transplantation, cold stress–induced cellular injury is a significant problem in clinical orthotopic liver transplantation (OLT). Because a recent study showed that cold stress activates ferroptosis, a form of regulated cell death, we investigated whether and how ferroptosis determines OLT outcomes in mice and humans. Treatment with ferroptosis inhibitor (ferrostatin-1) during cold preservation reduced lipid peroxidation (malondialdehyde; MDA), primarily in liver sinusoidal endothelial cells (LSECs), and alleviated ischemia/reperfusion injury in mouse OLT. Similarly, ferrostatin-1 reduced cell death in cold-stressed LSEC cultures. LSECs deficient in nuclear factor erythroid 2-related factor 2 (NRF2), a critical regulator of ferroptosis, were susceptible to cold stress–induced cell death, concomitant with enhanced endoplasmic reticulum (ER) stress and expression of mitochondrial Ca2+ uptake regulator (MICU1). Indeed, supplementing MICU1 inhibitor reduced ER stress, MDA expression, and cell death in NRF2-deficient but not WT LSECs, suggesting NRF2 is a critical regulator of MICU1-mediated ferroptosis. Consistent with murine data, enhanced liver NRF2 expression reduced MDA levels, hepatocellular damage, and incidence of early allograft dysfunction in human OLT recipients. This translational study provides a clinically applicable strategy in which inhibition of ferroptosis during liver cold preservation mitigates OLT injury by protecting LSECs from peritransplant stress via an NRF2-regulatory mechanism.
Hidenobu Kojima, Hirofumi Hirao, Kentaro Kadono, Takahiro Ito, Siyuan Yao, Taylor Torgerson, Kenneth J. Dery, Hiroaki Kitajima, Takahiro Ogawa, Fady M. Kaldas, Douglas G. Farmer, Jerzy W. Kupiec-Weglinski
The human adult immune system maintains normal T-cell counts and compensates for T-cell loss over lifetime mainly through peripheral homeostatic proliferation after the ability of the thymus to generate new T cells has rapidly declined at adolescence. This process is mainly driven by STAT5-activating cytokines, most importantly IL-7, and is very effective in maintaining a large naïve CD4 T cell compartment into older age. Here, we describe that naïve CD4 T cells undergo adaptations to optimize IL-7 responses by upregulating the guanine-nucleotide exchange factor PREX1 at older age. PREX1 promotes nuclear translocation of phosphorylated STAT5, thereby supporting homeostatic proliferation in response to IL-7. Through the same mechanism, increased expression of PREX1 also biases naïve cells to differentiate into effector T cells. These findings are consistent with the concept that primarily beneficial adaptations during aging, i.e., improved homeostasis, account for unfavorable functions of the aged immune system, in this case biased differentiation.
Huimin Zhang, Hirohisa Okuyama, Abhinav Jain, Rohit R. Jadhav, Bowen Wu, Ines Sturmlechner, Jose Morales, Shozo Ohtsuki, Cornelia M. Weyand, Jörg J. Goronzy
Inappropriate immune activity is key in the pathogenesis of multiple diseases and is typically driven by excess inflammation and/or autoimmunity. IL-1 is often the effector due to its powerful role in both innate and adaptive immunity, and thus is tightly controlled at multiple levels. IL-1R2 antagonises IL-1, but effects of losing this regulation is unknown. We find IL-1R2 resolves inflammation by rapidly scavenging free IL-1. Specific IL-1R2 loss in germinal centre (GC) T follicular regulatory (Tfr) cells increases the GC response after a first, but not booster, immunisation, with more T follicular helper (Tfh) cells, GC B cells and antigen-specific antibodies, which is reversed upon IL-1 blockade. However, IL-1 signalling is not obligate for GC reactions, as wildtype and Il1r1–/– mice show equivalent phenotypes, suggesting GC IL-1 is normally restrained by IL-1R2. Fascinatingly, germline Il1r2–/– mice do not show this phenotype, but conditional Il1r2 deletion in adulthood recapitulates it, implying compensation during development counteracts IL-1R2 loss. Finally, patients with ulcerative colitis or Crohn’s disease have lower serum IL-1R2. Together, we show that IL-1R2 controls important aspects of innate and adaptive immunity, and that IL-1R2 level may contribute to human disease propensity and/or progression.
Katerina Pyrillou, Melanie Humphry, Lauren A. Kitt, Amanda Rodgers, Meritxell Nus, Martin R. Bennett, Kenneth G.C. Smith, Paul A. Lyons, Ziad Mallat, Murray C.H. Clarke
Atopic dermatitis (AD) is a persistent skin disease typified by symptoms of dry skin and recurrent eczema. AD patients are at heightened risk for Staphylococcus aureus (S. aureus) infection. Group 2 innate lymphoid cells (ILC2s) are mainly activated by epithelial cell-derived cytokines IL-33 and involved in the pathogenesis of AD. However, little is known about the effect of skin delipidization on the epithelial cell-derived cytokines and dermal ILC2s in AD. In our study, we investigated the mechanism by which S. aureus infection modulates and exacerbates the pathogenesis of dry skin, leading to type 2 inflammation in the context of innate immunity. In vivo, we found that S. aureus infection aggravated delipidization-induced dermal IL-33 release and dermal ILC2 accumulation, which exacerbated skin inflammation. We also noticed that Il33f/fK14cre mice and Tlr2–/– mice exhibited attenuated skin inflammation. In vitro, treatment with necroptosis inhibitors reduced IL-33 release from S. aureus-infected keratinocytes. Mechanistically, we observed an increase in the necroptosis-associated kinases, MLKL and RIPK3, in S. aureus-infected mice, indicating that IL-33 release was associated with necroptotic cell death responses. Our results reveal that S. aureus infection-elicited keratinocyte necroptosis contributes to IL-33-mediated type 2 inflammation, which exacerbates the pathogenesis of dry skin.
Chia-Hui Luo, Alan Chuan-Ying Lai, Chun-Chou Tsai, Wei-Yu Chen, Yu-Shan Chang, Ethan Ja-Chen Chung, Ya-Jen Chang
Pattern-Recognition Receptor responses are profoundly attenuated before the third trimester of gestation, in the relatively low oxygen human fetal environment. However, the mechanisms regulating these responses are uncharacterized. Herein, genome-wide transcription and functional metabolic experiments in primary neonatal monocytes linked the negative mTOR regulator DDIT4L to metabolic stress, cellular bioenergetics and innate immune activity. Using genetically engineered monocytic U937 cells, we confirmed that DDIT4L overexpression altered mitochondrial dynamics, suppressing their activity, and blunted LPS-induced cytokine responses. We also showed that monocyte mitochondrial function is more restrictive in earlier gestation, resembling the phenotype of DDIT4L-overexpressing U937 cells. Gene expression analyses in neonatal granulocytes, and lung macrophages in preterm infants confirmed upregulation of the DDIT4L gene in the early postnatal period, and also suggested a potential protective role against inflammation-associated chronic neonatal lung disease. Together, these data show that DDIT4L regulates mitochondrial activity and provide the first direct evidence for its potential role regulating innate immune activity in myeloid cells during development.
Christina Michalski, Claire Cheung, Ju Hee Oh, Emma Ackermann, Constantin R. Popescu, Anne-Sophie Archambault, Martin A. Prusinkiewicz, Rachel Da Silva, Abdelilah Majdoubi, Marina Viñeta Paramo, Rui Yang Xu, Frederic Reicherz, Annette E. Patterson, Liam Golding, Ashish A. Sharma, Chinten J. Lim, Paul C. Orban, Ramon I. Klein Geltink, Pascal M. Lavoie
Acute bacterial orchitis (AO) is a prevalent cause of intra-scrotal inflammation, often resulting in sub- or infertility. A frequent cause eliciting AO is uropathogenic Escherichia coli (UPEC), a gram negative pathovar, characterized by the expression of various iron acquisition systems to survive in a low-iron environment. On the host side, iron is tightly regulated by iron regulatory proteins (IRP) 1 and 2 and these factors are reported to play a role in testicular and immune cell function, however, their precise role remains unclear. Here, we showed in a mouse model of UPEC-induced orchitis that the absence of IRP1 results in reduced immune response and testicular damage. Compared to infected wild-type (WT)-mice, testis of UPEC-infected Irp1–/– mice showed impaired ERK signaling. Conversely, IRP2 deletion led to a stronger inflammatory response. Notably, differences in immune cell infiltrations were observed among the different genotypes. In contrast to WT and Irp2–/– mice, no increase in monocytes and neutrophils was detected in testis of Irp1–/– mice upon UPEC-infection. Interestingly, in Irp1–/– UPEC-infected testis, we observed an increase in a subpopulation of macrophages (F4/80+ CD206+) associated with anti-inflammatory and wound-healing activities compared to WT. These findings suggest that IRP1 deletion may protect against UPEC-induced inflammation by modulating ERK signaling and dampening the immune response.
Niraj Ghatpande, Aileen Harrer, Bar Azoulay-Botzer, Noga Guttmann-Raviv, Sudhanshu Bhushan, Andreas Meinhardt, Esther G. Meyron-Holtz
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