Immunology
Abstract
Pre-existing humoral immunity to recombinant adeno-associated viral (AAV) vectors restricts the treatable patient population and efficacy of human gene therapies. Approaches to clear neutralizing antibodies (NAbs), such as plasmapheresis and immunosuppression are either ineffective or cause undesirable side effects. Here, we describe a clinically relevant strategy to rapidly and transiently degrade NAbs prior to AAV administration using an IgG degrading enzyme (IdeZ). We demonstrate that recombinant IdeZ efficiently cleaves IgG in dog, monkey and human antisera. Prophylactically administered IdeZ cleaves circulating, human IgG in mice and prevents AAV neutralization in vivo. In macaques, a single intravenous dose of IdeZ rescues AAV transduction by transiently reversing seropositivity. Importantly, IdeZ efficiently cleaves NAbs and rescues AAV transduction in mice passively immunized with individual human donor sera representing a diverse population. Our antibody clearance approach presents a new paradigm for expanding the prospective patient cohort and improving efficacy of AAV gene therapy.
Authors
Zachary C. Elmore, Daniel K. Oh, Katherine E. Simon, Marco M. Fanous, Aravind Asokan
Abstract
Fibroblast-like synoviocytes (FLSs) are critical to joint inflammation and destruction in rheumatoid arthritis (RA). Increased glycolysis in RA FLSs contributes to persistent joint damage. SUMOylation, a posttranslational modification of proteins, plays an important role in initiation and development of many diseases. However, the role of small ubiquitin-like modifier–activating (SUMO-activating) enzyme 1 (SAE1)/ubiquitin like modifier activating enzyme 2 (UBA2) in regulating the pathogenic FLS behaviors is unknown. Here, we found an increased expression of SAE1 and UBA2 in FLSs and synovial tissues from patients with RA. SAE1 or UBA2 knockdown by siRNA and treatment with GA, an inhibitor of SAE1/UBA2-mediated SUMOylation, resulted in reduced glycolysis, aggressive phenotype, and inflammation. SAE1/UBA2-mediated SUMOylation of pyruvate kinase M2 (PKM2) promoted its phosphorylation and nuclear translocation and decreased PK activity. Moreover, inhibition of PKM2 phosphorylation increased PK activity and suppressed glycolysis, aggressive phenotype, and inflammation. We further demonstrated that STAT5A mediated SUMOylated PKM2-induced glycolysis and biological behaviors. Interestingly, GA treatment attenuated the severity of arthritis in mice with collagen-induced arthritis and human TNF-α transgenic mice. These findings suggest that an increase in synovial SAE1/UBA2 may contribute to synovial glycolysis and joint inflammation in RA and that targeting SAE1/UBA2 may have therapeutic potential in patients with RA.
Authors
Cuicui Wang, Youjun Xiao, Minxi Lao, Jingnan Wang, Siqi Xu, Ruiru Li, Xuanxian Xu, Yu Kuang, Maohua Shi, Yaoyao Zou, Qingwen Wang, Liuqin Liang, Song Guo Zheng, Hanshi Xu
Abstract
Impaired tolerance to innocuous particles during allergic asthma has been linked to the increased plasticity of FoxP3+ regulatory T (Treg) cells, reprogramming into pathogenic effector cells, thus exacerbating airway disease. Failure in tolerance is suggested to be driven by TH2 inflammatory signals. The canonical IL-4Rα-signalling, an essential driver of TH2-type airway responses to allergens was investigated on its in vivo role on the regulatory function of FoxP3+ Tregs in allergic asthma. We used transgenic Foxp3creIL-4rα-/lox and littermate control mice to investigate the role of IL-4/IL-13 signalling via T regs in a house dust mite (HDM)-induced allergic airway disease. We sensitised mice intratracheally on day 0 and challenged them on day 6-10 and analysed airway hyperresponsiveness (AHR), airway inflammation, mucus production and cellular profile on day 14. In the absence of IL-4Rα responsiveness on FoxP3+ Tregs, there was an exacerbated AHR and airway inflammation in HDM-sensitised mice. Interestingly, a reduced induction of FoxP3+ Tregs accompanied increased IL-33 “alarmin” production and innate lymphoid cells type 2 (ILC2) activation in the lung exacerbating airway hyperreactivity and lung eosinophilia. We conclude that IL-4Rα unresponsive FoxP3+ T regulatory cells results in exaggerated innate TH2-type, IL-33-dependent airway inflammation and a break in tolerance during allergic asthma.
Authors
Jermaine Khumalo, Frank Kirstein, Sabelo Hadebe, Frank Brombacher
Abstract
Protein phosphatase 2A is a ubiquitously expressed serine/threonine phosphatase which comprises a scaffold, a catalytic and multiple regulatory subunits and has been shown to be important in the expression of autoimmunity. We considered that a distinct subunit may account for the decreased production of interleukin-2 (IL-2) in people and mice with systemic autoimmunity. We show that the regulatory subunit PPP2R2D is increased in T cells from people with systemic lupus erythematosus and regulates IL-2 production. Mice lacking PPP2R2D only in T cells produce more IL-2 because the IL-2 gene and genes coding for IL-2 enhancing transcription factors remain open and the levels of the enhancer phosphorylated CREB are high. Mice with T cell-specific PPP2R2D deficiency display less systemic autoimmunity when exposed to a TLR7 stimulator. While genes related to regulatory T cell function do not change in the absence of PPP2R2D, regulatory T cells exhibit high suppressive function in vitro and in vivo. Because the ubiquitous expression of protein phosphatase 2A cannot permit systemic therapeutic manipulation, the identification of regulatory subunits able to control specific T cell functions opens the way for the development of novel, function-specific drugs.
Authors
Wenliang Pan, Amir Sharabi, Andrew P. Ferretti, Yinfeng Zhang, Catalina Burbano, Nobuya Yoshida, Maria G. Tsokos, George C. Tsokos
Abstract
Identification of MHC class I bound peptides by immunopurification of MHC complexes and subsequent analysis by mass spectrometry is crucial for understanding T cell immunology and immunotherapy. Investigation of the steps for the MHC ligand isolation process revealed biases in widely used isolation techniques towards peptides of lower hydrophobicity. As MHC ligand hydrophobicity correlates positively with immunogenicity, identification of more hydrophobic MHC ligands could potentially lead to more effective isolation of immunogenic peptides as targets for immunotherapies. We solved this problem by use of higher concentrations of acetonitrile (ACN) for the separation of MHC ligands and their respective complexes. This increased overall MHC ligand identifications by 2-fold, detection of cancer germline antigen-derived peptides by 50%, and resulted in profound variations in isolation efficacy between different MHC alleles correlating with the hydrophobicity of their anchor residues. Overall, these insights enabled a more complete view on the immunopeptidome and overcame a systematic underrepresentation of these critical MHC ligands of high hydrophobicity.
Authors
Martin G. Klatt, Kyeara N. Mack, Yang Bai, Zita E.H. Aretz, Levy I. Nathan, Sung Soo Mun, Tao Dao, David A. Scheinberg
Abstract
In prior studies, we delineated the landscape of neoantigens arising from nonsynonymous point mutations in a murine pancreatic cancer model, Panc02. We developed a peptide vaccine by targeting neoantigens predicted using a pipeline that incorporates the MHC binding algorithm NetMHC. The vaccine, when combined with immune checkpoint modulators, elicited a robust neoepitope-specific antitumor immune response and led to tumor clearance. However, only a small fraction of the predicted neoepitopes induced T cell immunity, similarly to that reported for neoantigen vaccines tested in clinical studies. While these studies have used binding affinities to MHC I as surrogates for T cell immunity, this approach does not include spatial information on the mutated residue that is crucial for TCR activation. Here, we investigate conformational alterations in and around the MHC binding groove induced by selected minimal neoepitopes, and we examine the influence of a given mutated residue as a function of its spatial position. We found that structural parameters, including the solvent-accessible surface area (SASA) of the neoepitope and the position and spatial configuration of the mutated residue within the sequence, can be used to improve the prediction of immunogenic neoepitopes for inclusion in cancer vaccines.
Authors
Neeha Zaidi, Mariya Soban, Fangluo Chen, Heather Kinkead, Jocelyn Mathew, Mark Yarchoan, Todd D. Armstrong, Shozeb Haider, Elizabeth M. Jaffee
Abstract
Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin (Ig)-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress for this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAb) was generated. LILBR3-specific mAb bound to discrete epitopes in either Ig-like domain two or four. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor.
Authors
Muchaala J. Yeboah, Charys Papagregoriou, Des C. Jones, H. T. Claude Chan, Guangan Hu, Justine S. McPartlan, Torbjörn Schiött, Ulrika T. Mattson, C. Ian Mockridge, Ulla-Carin Tornberg, Björn Hambe, Anne Ljungars, Mikael Mattsson, Ivo Tews, Martin J. Glennie, Stephen M. Thirdborough, John Trowsdale, Björn Frendéus, Jianzhu Chen, Mark S. Cragg, Ali Roghanian
Abstract
Hidradenitis Suppurativa (HS) is a debilitating chronic inflammatory skin disease characterized by chronic abscess formation and development of multiple draining sinus tracts in the groin, axillae, and perineum. Utilizing proteomic and transcriptomic approaches, we characterized the inflammatory responses in HS in depth, revealing immune responses centered around IFN-γ, IL-36, and TNF, with lesser contribution from IL-17A. We further identified B cells and plasma cells, with associated increases in immunoglobulin production and complement activation, as pivotal players in HS pathogenesis, with BTK and SYK pathway activation as a central signal transduction networks in HS. These data provide preclinical evidence to accelerate the path towards clinical trials targeting BTK and SYK signaling in moderate to severe HS.
Authors
Johann E. Gudjonsson, Lam C. Tsoi, Feiyang Ma, Allison C. Billi, Kelsey R. van Straalen, Allard R.J.V. Vossen, H.H. Zee, Paul W. Harms, Rachael Wasikowski, Christine M. Yee, Syed Monem Rizvi, Xianying Xing, Enze Xing, Olesya Plazyo, Chang Zeng, Matthew T. Patrick, Margaret M. Lowe, Richard E. Burney, Jeffrey H. Kozlow, Jill R. Cherry-Bukowiec, Yanyun Jiang, Joseph Kirma, Stephan Weidinger, Kelly C. Cushing, Michael D. Rosenblum, Celine C. Berthier, Amanda S. MacLeod, John J. Voorhees, Fei Wen, J. Michelle Kahlenberg, Emanual Maverakis, Robert L. Modlin, Errol P. Prens
Abstract
African green monkeys (AGMs) are natural hosts of Simian immunodeficiency virus (SIV) that post-thymically down-regulate CD4 to maintain a large population of CD4-CD8aa+ virus-resistant cells with T-helper functionality, which can result in AGMs becoming apparently cured of SIVagm infection. To understand the mechanisms of this process we performed genome-wide transcriptional analysis on T cells induced to down-regulate CD4 in vitro from AGMs and closely-related Patas monkeys, and T cells that maintain CD4 expression from rhesus macaques. In T cells that down-regulated CD4, pathway analysis revealed an atypical regulation ofthe DNA methylation machinery, which was reversible when pharmacologically targeted with 5-aza-2deoxycytidine. This signature was driven largely by the dioxygenase TET3 that became down-regulated with loss of CD4 expression. CpG motifs within the AGM CD4 promoter region became methylated during CD4 downregulation in vitro and were stably imprinted in AGM CD4-CD8aa+ T cells sorted directly ex vivo. These results suggest AGMs employ epigenetic mechanisms to durably silence the CD4 gene. Manipulation of these mechanisms could provide avenues for modulating SIV and human immunodeficiency virus (HIV)-1 entry receptor expression in hosts that become progressively SIV-infected, which could lead to novel therapeutic interventions aimed to reduce HIV viremia in vivo.
Authors
Joseph C. Mudd, Stephen Lai, Sanjana Shah, Andrew R. Rahmberg, Jacob K. Flynn, Carly E. Starke, Molly R. Perkins, Amy Ransier, Samuel Darko, Daniel Douek, Vanessa Hirsch, Mark J. Cameron, Jason M. Brenchley
Abstract
Myeloid cells orchestrate the anti-tumor immune response and influence the efficacy of immune checkpoint blockade (ICB) therapies. We and others have previously shown that interleukin 32 (IL-32) mediates dendritic cell (DC) differentiation and macrophage activation. Here, we demonstrate that IL-32 expression in human melanoma positively correlates with overall survival, response to ICB, and an immune inflamed tumor microenvironment (TME) enriched in mature DC, M1 macrophages and CD8+ T cells. Treatment of B16F10 murine melanomas with IL-32 increased the frequencies of activated, tumor-specific CD8+ T cells, leading to the induction of systemic tumor immunity. Our mechanistic in vivo studies revealed a novel role of IL-32 in activating intra-tumoral DC and macrophages to act in concert to prime CD8+ T cells and recruit them into the TME through CCL5. Thereby, IL-32 treatment reduced tumor growth and rendered ICB resistant B16F10 tumors responsive to anti-PD-1 therapy without toxicity. Furthermore, increased baseline IL-32 gene expression was associated with response to nivolumab and pembrolizumab in two independent human melanoma patient cohorts, implying IL-32 as a predictive biomarker for anti-PD-1 therapy. Collectively, this study suggests IL-32 as a potent adjuvant in immunotherapy to enhance the efficacy of ICB to patients with non-T cell inflamed TME.
Authors
Thomas Gruber, Mirela Kremenovic, Hassan Sadozai, Nives Rombini, Lukas Baeriswyl, Fabienne Maibach, Robert L. Modlin, Michel Gilliet, Diego Von Werdt, Robert E. Hunger, Giulia Parisi, Gabriel Abril-Rodriguez, Antoni Ribas, Mirjam Schenk
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