Immunology
Abstract
Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents.
Authors
Christopher J. Holland, Rory M. Crean, Johanne M. Pentier, Ben de Wet, Angharad Lloyd, Velupillai Srikannathasan, Nikolai Lissin, Katy A. Lloyd, Thomas H. Blicher, Paul J. Conroy, Miriam Hock, Robert J. Pengelly, Thomas E. Spinner, Brian Cameron, Elizabeth A. Potter, Anitha Jeyanthan, Peter E. Molloy, Malkit Sami, Milos Aleksic, Nathaniel Liddy, Ross A. Robinson, Stephen Harper, Marco Lepore, Chris R. Pudney, Marc W. van der Kamp, Pierre J. Rizkallah, Bent K. Jakobsen, Annelise Vuidepot, David K. Cole
Abstract
Microbial ingestion by a macrophage results in the formation of an acidic phagolysosome but the host cell has no information on the pH susceptibility of the ingested organism. This poses a problem for the macrophage and raises the fundamental question of how the phagocytic cell optimizes the acidification process to prevail. We analyzed the dynamical distribution of phagolysosomal pH in murine and human macrophages that had ingested live or dead Cryptococcus neoformans cells, or inert beads. Phagolysosomal acidification produced a range of pH values that approximated normal distributions, but these differed from normality depending on ingested particle type. Analysis of the increments of pH reduction revealed no forbidden ordinal patterns, implying that phagosomal acidification process was a stochastic dynamical system. Using simulation modeling, we determined that by stochastically acidifying a phagolysosome to a pH within the observed distribution, macrophages sacrificed a small amount of overall fitness to reduce their overall variation in fitness. Hence, chance in the final phagosomal pH introduces unpredictability to the outcome of the macrophage-microbe, which implies a bet-hedging strategy that benefits the macrophage. While bet hedging is common in biological systems at the organism level, our results show its use at the organelle and cellular level.
Authors
Quigly Dragotakes, Kaitlin M. Stouffer, Man Shun Fu, Yehonatan Sella, Christine Youn, Olivia Insun Yoon, Carlos M. De Leon-Rodriguez, Joudeh Freij, Aviv Bergman, Arturo Casadevall
Abstract
IL-17–producing RORγt+ γδ T cells (γδT17 cells) are innate lymphocytes that participate in type 3 immune responses during infection and inflammation. Herein, we show that γδT17 cells rapidly proliferate within neonatal lymph nodes and gut, where, upon entry, they upregulate T-bet and coexpress IL-17, IL-22, and IFN-γ in a STAT3- and retinoic acid–dependent manner. Neonatal expansion was halted in mice conditionally deficient in STAT5, and its loss resulted in γδT17 cell depletion from all adult organs. Hyperactive STAT5 mutant mice showed that the STAT5A homolog had a dominant role over STAT5B in promoting γδT17 cell expansion and downregulating gut-associated T-bet. In contrast, STAT5B preferentially expanded IFN-γ–producing γδ populations, implying a previously unknown differential role of STAT5 gene products in lymphocyte lineage regulation. Importantly, mice lacking γδT17 cells as a result of STAT5 deficiency displayed a profound resistance to experimental autoimmune encephalomyelitis. Our data identify that the neonatal microenvironment in combination with STAT5 is critical for post-thymic γδT17 development and tissue-specific imprinting, which is essential for infection and autoimmunity.
Authors
Darshana Kadekar, Rasmus Agerholm, John Rizk, Heidi A. Neubauer, Tobias Suske, Barbara Maurer, Monica Torrellas Viñals, Elena M. Comelli, Amel Taibi, Richard Moriggl, Vasileios Bekiaris
Abstract
Food allergies are a major clinical problem and are driven by IgE antibodies specific for food antigens. T follicular regulatory (TFR) cells are a specialized subset of Foxp3+ T cells that modulate antibody responses. Here we analyzed the role of TFR cells in regulating antigen-specific IgE using a peanut-based food allergy model in mice. Peanut-specific IgE titers and anaphylaxis responses were significantly blunted in TFR cell-deficient Foxp3-cre Bcl6-fl/fl mice. Loss of TFR cells led to greatly increased non-specific IgE levels, showing that TFR cells have both helper and suppressor functions on IgE production in the GC that work together to facilitate the production of antigen-specific IgE. Foxp3-cre Pten-fl/fl mice with augmented TFR cell responses had markedly higher levels of peanut-specific IgE, revealing an active helper function by TFR cells on antigen-specific IgE. The helper function of TFR cells for IgE production involves IL-10, and the loss of IL-10 signaling by B cells led to a severely curtailed peanut-specific IgE response, decreased GC B cell survival and loss of GC dark zone B cells after peanut sensitization. We thus reveal that TFR cells have an unexpected helper role in promoting food allergy and are a novel target for drug development.
Authors
Markus M. Xie, Qiang Chen, Hong Liu, Kai Yang, Byunghee Koh, Hao Wu, Soheila J. Maleki, Barry K. Hurlburt, Joan Cook-Mills, Mark H. Kaplan, Alexander L. Dent
Abstract
Allergic asthma is mediated by T helper 2 (Th2) responses to inhaled allergens. Although previous experiments indicated that Notch signaling activates expression of the key Th2 transcription factor Gata3, it remains controversial how Notch promotes allergic airway inflammation. Here we show that T cell-specific Notch deficiency in mice prevented house dust mite-driven eosinophilic airway inflammation and significantly reduced Th2 cytokine production, serum IgE levels and airway hyperreactivity. However, transgenic Gata3 overexpression in Notch-deficient T cells only partially rescued this phenotype. We found that Notch signaling was not required for T cell proliferation or Th2 polarization. Instead, Notch-deficient in vitro polarized Th2 cells showed reduced accumulation in the lungs upon in vivo transfer and allergen challenge, as Notch-deficient Th2 cells were retained in the lung draining lymph nodes. Transcriptome analyses and sequential adoptive transfer experiments revealed that while Notch-deficient lymph node Th2 cells established competence for lung migration, they failed to upregulate the sphingosine 1-phosphate receptor (S1PR1) and its critical upstream transcriptional activator Krüppel-like factor 2 (KLF2). As this KLF2-S1PR1 axis represents the essential cell-intrinsic regulator of T cell lymph node egress, we conclude that the druggable Notch signaling pathway licenses the Th2 response in allergic airway inflammation via promoting lymph node egress.
Authors
Irma Tindemans, Anne van Schoonhoven, Alex KleinJan, Marjolein J.W. de Bruijn, Melanie Lukkes, Menno van Nimwegen, Anouk van den Branden, Ingrid M. Bergen, Odilia B. J. Corneth, Wilfred F.J. van IJcken, Ralph Stadhouders, Rudi W. Hendriks
Abstract
A common variant in the RAB27A gene in adults was recently found to be associated with the fractional exhaled nitric oxide level, a marker of eosinophilic airway inflammation. The small GTPase, Rab27, is known to regulate intracellular vesicle traffic, although its role in allergic responses is unclear. We demonstrated that exophilin-5, a Rab27 binding protein, was predominantly expressed in both the major IL-33 producers, lung epithelial cells, and the specialized IL-5 and IL-13 producers in CD44highCXCR3lowCD62Llow pathogenic T helper 2 (Th2) cell population in mice. Exophilin-5 deficiency increased stimulant-dependent damages and IL-33 secretion of lung epithelial cells. Moreover, it enhanced IL-5 and IL-13 production in response to TCR and IL-33 stimulation from a specific subset of pathogenic Th2 cells that expresses a high level of IL-33 receptor, which exacerbated allergic airway inflammation in a mouse model of asthma. Mechanistically, exophilin-5 regulates extracellular superoxide release, intracellular ROS production, and phosphoinositide 3-kinase activity by controlling intracellular traffic of Nox2-containing vesicles, which seems to prevent the overactivation of pathogenic Th2 cells mediated by IL-33. This is the first report to establish the significance of Rab27-related protein exophilin-5 in the development of allergic airway inflammation, and provides new insights into the pathophysiology of asthma.
Authors
Katsuhide Okunishi, Hao Wang, Maho Suzukawa, Ray Ishizaki, Eri Kobayashi, Miho Kihara, Takaya Abe, Jun-ichi Miyazaki, Masafumi Horie, Akira Saito, Hirohisa Saito, Susumu Nakae, Tetsuro Izumi
Abstract
Hidradenitis suppurativa (HS) is a chronic, relapsing, inflammatory skin disease. HS appears to be a primary abnormality in the pilosebaceous-apocrine unit. In this work, we characterized hair follicle stem cells isolated from HS patients and more precisely the Outer Root Sheath Cells (ORS). We show that hair follicles from HS patients have an increased number of proliferating progenitor cells and lose quiescent stem cells. Remarkably, we also show that the progression of replication forks is altered in HS-ORS and activates the ATR-CHK1 pathway. These alterations are associated with an increased number of micronuclei and with the presence of cytoplasmic ssDNA, leading to the activation of IFI16-STING pathway and the production of type I IFNs. This mechanistic analysis of the etiology of HS in the hair follicle stem cells compartment establishes a formal link between the genetic predisposition and skin inflammation observed in HS.
Authors
Cindy Orvain, Yea-Lih Lin, Francette Jean-Louis, Hakim Hocini, Barbara Hersant, Yamina Bennasser, Nicolas Ortonne, Claire Hotz, Pierre Wolkenstein, Michele Boniotto, Pascaline Tisserand, Cecile Lefebvre, Jean-Daniel Lelievre, Monsef Benkirane, Philippe Pasero, Yves Levy, Sophie Hue
Abstract
Germinal center (GC) responses require B cells to respond to a dynamic set of intercellular and microenvironmental signals that instruct B cell positioning, differentiation, and metabolic reprogramming. ROCK2, a serine-threonine kinase that can be therapeutically targeted by ROCK inhibitors or statins, is a key downstream effector of RHOA-GTPases. While RHOA-mediated pathways are emerging as critical regulators of GC responses, the role of ROCK2 in B cells is unknown. Here, we find that ROCK2 was activated in response to key T cell signals like CD40 and IL21 and that it regulated GC formation and maintenance. RNA-seq analyses revealed that ROCK2 controlled a unique transcriptional program in GC B cells that promoted optimal GC polarization and cholesterol biosynthesis. ROCK2 regulated this program by restraining AKT activation and subsequently enhancing FOXO1 activity. ATAC-seq and biochemical analyses revealed that the effects of ROCK2 on cholesterol biosynthesis were instead mediated via a novel mechanism. ROCK2 directly phosphorylated IRF8, a crucial mediator of GC responses, and promoted its interaction with SREBP2 at key regulatory regions controlling the expression of cholesterol biosynthetic enzymes, resulting in optimal recruitment of SREBP2 at these sites. These findings thus uncover ROCK2 as a multifaceted and therapeutically targetable regulator of GC responses.
Authors
Edd Ricker, Yurii Chinenov, Tania Pannellini, Danny M. Flores Castro, Chao Ye, Sanjay Gupta, Michela Manni, James K. Liao, Alessandra Pernis
Abstract
BACKGROUND. Since December 2019, an outbreak of Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, and is now becoming a global threat. We aimed to delineate and compare the immunologic features of severe and moderate COVID-19. METHODS. In this retrospective study, the clinical and immunologic characteristics of 21 patients (17 male and 4 female) with COVID-19 were analyzed. These patients were classified as severe (11 cases) and moderate (10 cases) according to the Guidelines released by the National Health Commission of China. RESULTS. The median age of severe and moderate cases was 61.0 and 52.0 years, respectively. Common clinical manifestations included fever, cough and fatigue. Compared to moderate cases, severe cases more frequently had dyspnea, lymphopenia, and hypoalbuminemia, with higher levels of alanine aminotransferase, lactate dehydrogenase, C-reactive protein, ferritin and D-dimer as well as markedly higher levels of IL-2R, IL-6, IL-10, and TNF-α. Absolute number of T lymphocytes, CD4+T and CD8+T cells decreased in nearly all the patients, and were markedly lower in severe cases (294.0, 177.5 and 89.0 × 106/L) than moderate cases (640.5, 381.5 and 254.0 × 106/L). The expressions of IFN-γ by CD4+T cells tended to be lower in severe cases (14.1%) than moderate cases (22.8%). CONCLUSION. The SARS-CoV-2 infection may affect primarily T lymphocytes particularly CD4+T and CD8+ T cells, resulting in decrease in numbers as well as IFN-γ production. These potential immunological markers may be of importance due to their correlation with disease severity in COVID-19.
Authors
Guang Chen, Di Wu, Wei Guo, Yong Cao, Da Huang, Hongwu Wang, Tao Wang, Xiaoyun Zhang, Huilong Chen, Haijing Yu, Xiaoping Zhang, Minxia Zhang, Shiji Wu, Jianxin Song, Tao Chen, Meifang Han, Shusheng Li, Xiaoping Luo, Jianping Zhao, Qin Ning
Abstract
Alloantibodies in pre-sensitized transplant candidates deposit complement membrane attack complexes (MAC) on graft endothelial cells (ECs), increasing risk of CD8+ T cell-mediated acute rejection. We recently showed (a) human ECs endocytose MAC into Rab5+ endosomes, creating a signaling platform that stabilizes NF-κB–inducing kinase (NIK) protein; (b) endosomal NIK activates both non-canonical NF-κB signaling to synthesize pro-IL-1β and an NLRP3 inflammasome to process and secrete active IL-1β; and (c) IL-1β activates ECs, increasing recruitment and activation of alloreactive effector memory CD4+ T (TEM) cells. Here, we report IFN-γ priming induced nuclear expression of IL-15/IL-15Rα complexes in cultured human ECs and that MAC-induced IL-1β stimulated translocation of IL-15/IL-15Rα complexes to the EC surface in a canonical NF-κB-dependent process, where IL-15/IL-15Rα transpresentation increased activation and maturation of alloreactive CD8+ TEM. Blocking NLRP3 inflammasome assembly, IL-1 receptor or IL-15 on ECs inhibited the augmented CD8+ TEM responses, indicating this pathway was not redundant. Adoptively transferred alloantibody and mouse complement deposition induced IL-15/IL-15Rα expression by human ECs lining human coronary artery grafts in immunodeficient mice and enhanced intimal CD8+ T cell infiltration, which was markedly reduced by inflammasome inhibition, linking alloantibody to acute rejection. Inhibiting MAC signaling may similarly limit other complement-mediated pathologies.
Authors
Catherine B. Xie, Bo Jiang, Lingfeng Qin, George Tellides, Nancy C. Kirkiles-Smith, Dan Jane-wit, Jordan S. Pober
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)