Senescent cells (SnCs) are implicated in the pathogenesis of age-related diseases including osteoarthritis (OA), in part via expression of a senescence-associated secretory phenotype (SASP) that includes immunologically relevant factors and cytokines. In a model of posttraumatic OA (PTOA), anterior cruciate ligament transection (ACLT) induced a type 17 immune response in the articular compartment and draining inguinal lymph nodes (LNs) that paralleled expression of the senescence marker p16INK4a (Cdkn2a) and p21 (Cdkn1a). Innate lymphoid cells, γδ+ T cells, and CD4+ T cells contributed to IL-17 expression. Intra-articular injection of IL-17–neutralizing antibody reduced joint degeneration and decreased expression of the senescence marker Cdkn1a. Local and systemic senolysis was required to attenuate tissue damage in aged animals and was associated with decreased IL-17 and increased IL-4 expression in the articular joint and draining LNs. In vitro, we found that Th17 cells induced senescence in fibroblasts and that SnCs skewed naive T cells toward Th17 or Th1, depending on the presence of TGF-β. The SASP profile of the inflammation-induced SnCs included altered Wnt signaling, tissue remodeling, and cell-cycle pathways not previously implicated in senescence. These findings provide molecular targets and mechanisms for senescence induction and therapeutic strategies to support tissue healing in an aged environment.
Heather J. Faust, Hong Zhang, Jin Han, Matthew T. Wolf, Ok Hee Jeon, Kaitlyn Sadtler, Alexis N. Peña, Liam Chung, David R. Maestas Jr., Ada J. Tam, Drew M. Pardoll, Judith Campisi, Franck Housseau, Daohong Zhou, Clifton O. Bingham III, Jennifer H. Elisseeff
Late-onset inflammatory toxicities resembling hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS) occur after chimeric antigen receptor T cell (CAR T cell) infusion and represent a therapeutic challenge. Given the established link between perforin deficiency and primary HLH, we investigated the role of perforin in anti-CD19 CAR T cell efficacy and HLH-like toxicities in a syngeneic murine model. Perforin contributed to both CD8+ and CD4+ CAR T cell cytotoxicity but was not required for in vitro or in vivo leukemia clearance. Upon CAR-mediated in vitro activation, perforin-deficient CAR T cells produced higher amounts of proinflammatory cytokines compared with WT CAR T cells. Following in vivo clearance of leukemia, perforin-deficient CAR T cells reexpanded, resulting in splenomegaly with disruption of normal splenic architecture and the presence of hemophagocytes, which are findings reminiscent of HLH. Notably, a substantial fraction of patients who received anti-CD22 CAR T cells also experienced biphasic inflammation, with the second phase occurring after the resolution of cytokine release syndrome, resembling clinical manifestations of HLH. Elevated inflammatory cytokines such as IL-1β and IL-18 and concurrent late CAR T cell expansion characterized the HLH-like syndromes occurring in the murine model and in humans. Thus, a murine model of perforin-deficient CAR T cells recapitulated late-onset inflammatory toxicities occurring in human CAR T cell recipients, providing therapeutically relevant mechanistic insights.
Kazusa Ishii, Marie Pouzolles, Christopher D. Chien, Rebecca A. Erwin-Cohen, M. Eric Kohler, Haiying Qin, Haiyan Lei, Skyler Kuhn, Amanda K. Ombrello, Alina Dulau-Florea, Michael A. Eckhaus, Haneen Shalabi, Bonnie Yates, Daniel A. Lichtenstein, Valérie S. Zimmermann, Taisuke Kondo, Jack F. Shern, Howard A. Young, Naomi Taylor, Nirali N. Shah, Terry J. Fry
The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the urgent need for assays that detect protective levels of neutralizing antibodies. We studied the relationship between anti-spike ectodomain (ECD), anti-receptor binding domain (RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by two in vitro assays using convalescent plasma samples from 68 COVID-19 patients. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers and in vitro VN titer. The probability of a VN titer ≥160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment, was ≥80% when anti-RBD or anti-ECD titers were ≥1:1350. Of all donors, 37% lacked VN titers ≥160. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease VN or IgG titers. Analysis of 2,814 asymptomatic adults found 73 individuals with anti-ECD IgG titers of ≥1:50 and strong positive correlation with anti-RBD and VN titers. Fourteen of these individuals had VN titers ≥1:160, all of which had anti-RBD titer ≥1:1350. We conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titers of ≥1:1350 may provide critical information about protection against COVID-19 disease.
Eric Salazar, Suresh V. Kuchipudi, Paul A. Christensen, Todd Eagar, Xin Yi, Picheng Zhao, Zhicheng Jin, S. Wesley Long, Randall J. Olsen, Jian Chen, Brian Castillo, Christopher Leveque, Dalton Towers, Jason J. Lavinder, Jimmy Gollihar, Jose A. Cardona, Gregory C. Ippolito, Ruth H. Nissly, Ian Bird, Denver Greenawalt, Randall M. Rossi, Abhinay Gontu, Sreenidhi Srinivasan, Indira Poojary, Isabella M. Cattadori, Peter Hudson, Nicole M. Josleyn, Laura Prugar, Kathleen E. Huie, Andrew S. Herbert, David W. Bernard, John M. Dye, Vivek Kapur, James M. Musser
Chronic viral infections are often established by the exploitation of immune regulatory mechanisms that result in non-functional T cell responses. Viruses that establish persistent infections remain a serious threat to human health. Sphingosine kinase (SphK) 2 generates sphingosine 1-phosphate, which is a molecule known to regulate multiple cellular processes. However, little is known about SphK2’s role during the host immune responses to viral infection. Here, we demonstrate that SphK2 functions during lymphocytic choriomeningitis virus Cl 13 (LCMV Cl 13) infection to limit T cell immune pathology, which subsequently aids in the establishment of virus-induced immunosuppression and the resultant viral persistence. The infection of Sphk2-deficient (Sphk2-/-) mice with LCMV Cl 13 led to the development of nephropathy and mortality via T cell-mediated immunopathology. Following LCMV infection, Sphk2-/- CD4+ T cells displayed increased activity and proliferation, and these cells promoted overactive LCMV Cl 13-specific CD8+ T cell responses. Notably, oral instillation of an SphK2-selective inhibitor promoted protective T cell responses and accelerated the termination of LCMV Cl 13 persistence in mice. Thus, SphK2 is indicated as an immunotherapeutic target for the control of persistent viral infections.
Caleb J. Studstill, Curtis J. Pritzl, Young-Jin Seo, Dae Young Kim, Chuan Xia, Jennifer J. Wolf, Ravi Nistala, Madhuvanthi Vijayan, Yong-Bin Cho, Kyung Won Kang, Sang-Myeong Lee, Bumsuk Hahm
Regulatory T (Treg) cells require Foxp3 expression and induction of a specific DNA hypomethylation signature during development, after which Treg cells persist as a self-renewing population that regulates immune system activation. Whether maintenance DNA methylation is required for Treg cell lineage development and stability and how methylation patterns are maintained during lineage self-renewal remain unclear. Here, we demonstrate that the epigenetic regulator Uhrf1 is essential for maintenance of methyl-DNA marks that stabilize Treg cellular identity by repressing effector T cell transcriptional programs. Constitutive and induced deficiency of Uhrf1 within Foxp3+ cells resulted in global yet non-uniform loss of DNA methylation, derepression of inflammatory transcriptional programs, destabilization of the Treg cell lineage, and spontaneous inflammation. These findings support a paradigm in which maintenance DNA methylation is required in distinct regions of the Treg cell genome for both lineage establishment and stability of identity and suppressive function.
Kathryn A. Helmin, Luisa Morales-Nebreda, Manuel A. Torres Acosta, Kishore R. Anekalla, Shang-Yang Chen, Hiam Abdala-Valencia, Yuliya Politanska, Paul Cheresh, Mahzad Akbarpour, Elizabeth M. Steinert, Samuel E. Weinberg, Benjamin D. Singer
The transcription factor interferon regulatory factor 5 (IRF5) is a central mediator of innate and adaptive immunity. Genetic variations within IRF5 associate with risk of systemic lupus erythematosus (SLE) and mice lacking Irf5 are protected from lupus onset and severity, but how IRF5 functions in the context of SLE disease progression remains unclear. Using the NZB/W F1 model of murine lupus, we show that murine Irf5 becomes hyper-activated before clinical onset. In SLE patients, IRF5 hyper-activation correlated with dsDNA titers. To test whether IRF5 hyper-activation is a targetable function, we developed novel inhibitors that are cell permeable, non-toxic and selectively bind to the inactive IRF5 monomer. Preclinical treatment of NZB/W F1 mice with inhibitor attenuated lupus pathology by reducing serum ANA, dsDNA titers and the number of circulating plasma cells, which alleviated kidney pathology and improved survival. Clinical treatment of MRL/lpr and pristane-induced mice with inhibitor led to significant reductions in dsDNA levels and improved survival. In ex vivo human studies, the inhibitor blocked SLE serum-induced IRF5 activation in healthy immune cells and reversed basal IRF5 hyper-activation in SLE immune cells. Altogether, this study provides the first in vivo clinical support for treating SLE patients with an IRF5 inhibitor.
Su Song, Saurav De, Victoria Nelson, Samin Chopra, Margaret LaPan, Kyle Kampta, Shan Sun, Mingzhu He, Cherrie D. Thompson, Dan Li, Tiffany Shih, Natalie Tan, Yousef Al-Abed, Eugenio Capitle, Cynthia Aranow, Meggan Mackay, William L. Clapp, Betsy J. Barnes
Human natural killer cell deficiency (NKD) arises from inborn errors of immunity that lead to impaired NK cell development, function, or both. Through the understanding of the biological perturbations in individuals with NKD, requirements for the generation of terminally mature functional innate effector cells can be elucidated. Here, we report a cause of NKD resulting from compound heterozygous mutations in minichromosomal maintenance complex member 10 (MCM10) that impaired NK cell maturation in a child with fatal susceptibility to CMV. MCM10 has not been previously associated with monogenic disease and plays a critical role in the activation and function of the eukaryotic DNA replisome. Through evaluation of patient primary fibroblasts, modeling patient mutations in fibroblast cell lines, and MCM10 knockdown in human NK cell lines, we have shown that loss of MCM10 function leads to impaired cell cycle progression and induction of DNA damage–response pathways. By modeling MCM10 deficiency in primary NK cell precursors, including patient-derived induced pluripotent stem cells, we further demonstrated that MCM10 is required for NK cell terminal maturation and acquisition of immunological system function. Together, these data define MCM10 as an NKD gene and provide biological insight into the requirement for the DNA replisome in human NK cell maturation and function.
Emily M. Mace, Silke Paust, Matilde I. Conte, Ryan M. Baxley, Megan M. Schmit, Sagar L. Patil, Nicole C. Guilz, Malini Mukherjee, Ashley E. Pezzi, Jolanta Chmielowiec, Swetha Tatineni, Ivan K. Chinn, Zeynep Coban Akdemir, Shalini N. Jhangiani, Donna M. Muzny, Asbjørg Stray-Pedersen, Rachel E. Bradley, Mo Moody, Philip P. Connor, Adrian G. Heaps, Colin Steward, Pinaki P. Banerjee, Richard A. Gibbs, Malgorzata Borowiak, James R. Lupski, Stephen Jolles, Anja K. Bielinsky, Jordan S. Orange
Background: Marked progress is achieved in understanding the physiopathology of COVID-19 that caused global pandemics. However, CD4+ T cell population that is critical for antibody response in COVID-19 is poorly understood. Methods: In this study, we provided a comprehensive analysis of peripheral CD4+ T cells of 13 COVID-19 convalescent patients, as defined as confirmed free of SARS-CoV-2 for 2-4 weeks, using flow cytometry, magnetic chemiluminescence enzyme antibody immunoassay and correlated the data with clinical characteristics. Results: We observed that relative to healthy individuals, convalescent patients displayed an altered peripheral CD4+ T cell spectrum. Specifically, consistent with other viral infections, cTFH1 cell associated with SARS-CoV-2 targeting antibodies, which was found to skew with disease severity as more severe individuals showed higher frequency of TEM and TFH-EM cells but a lower frequency of TCM, TFH-CM and TNaive cells, relative to mild and moderate patients. Interestingly, higher frequency of cTFH-EM cells correlated with lower number of recorded admission blood oxygen level in convalescent patients. These observations might constitute residual effects by which COVID-19 can impact the homeostasis of CD4+ T cells in the long-term and explain the highest ratio of class-switched virus-specific antibody producing individuals found in our severe COVID-19 cohort. Conclusion: Together, our study demonstrated close connection between CD4+ T cells and antibody production in COVID-19 convalescents.Funding: This study was supported by Six Talent Peaks Project in Jiangsu Province and the National Natural Science Foundation of China (NSFC) grants 81970759.
Fang Gong, Yaping Dai, Ting Zheng, Liang Cheng, Dan Zhao, Hao Wang, Min Liu, Hao Pei, Tengchuan Jin, Di Yu, Pengcheng Zhou
Proteins created from recurrent fusion genes like CBFB-MYH11 are prevalent in acute myeloid leukemia (AML), often necessary for leukemogenesis, persistent throughout the disease course, and highly leukemia specific, making them attractive neoantigen targets for immunotherapy. A nonameric peptide derived from a prevalent CBFB-MYH11 fusion protein was found to be immunogenic in HLA-B*40:01+ donors. High-avidity CD8+ T cell clones isolated from healthy donors killed CBFB-MYH11+ HLA-B*40:01+ AML cell lines and primary human AML samples in vitro. CBFB-MYH11–specific T cells also controlled CBFB-MYH11+ HLA-B*40:01+ AML in vivo in a patient-derived murine xenograft model. High-avidity CBFB-MYH11 epitope–specific T cell receptors (TCRs) transduced into CD8+ T cells conferred antileukemic activity in vitro. Our data indicate that the CBFB-MYH11 fusion neoantigen is naturally presented on AML blasts and enables T cell recognition and killing of AML. We provide proof of principle for immunologically targeting AML-initiating fusions and demonstrate that targeting neoantigens has clinical relevance even in low–mutational frequency cancers like fusion-driven AML. This work also represents a first critical step toward the development of TCR T cell immunotherapy targeting fusion gene–driven AML.
Melinda A. Biernacki, Kimberly A. Foster, Kyle B. Woodward, Michael E. Coon, Carrie Cummings, Tanya M. Cunningham, Robson G. Dossa, Michelle Brault, Jamie Stokke, Tayla M. Olsen, Kelda Gardner, Elihu Estey, Soheil Meshinchi, Anthony Rongvaux, Marie Bleakley
Background: Patients with diffuse midline gliomas (DMG), including diffuse intrinsic pontine glioma (DIPG), have dismal outcomes. We previously described the H3.3K27M mutation as a shared neoantigen in HLA-A*02.01+ H3.3K27M+ DMGs. Within the Pacific Pediatric Neuro-Oncology Consortium, we assessed safety and efficacy of an H3.3K27M-targeted peptide vaccine. Patients and Methods: Newly diagnosed patients aged 3-21 years with HLA-A*02.01+ and H3.3K27M+ status were enrolled into Stratum A (DIPG) and Stratum B (non-pontine DMG). Vaccine was administered in combination with poly-ICLC every three weeks for eight cycles, followed by once every six weeks. Immuno-monitoring and imaging occurred every three months. Imaging was centrally reviewed. Immunological responses were assessed in peripheral blood mononuclear cells using mass cytometry. Results: 19 patients enrolled in Stratum A (median age=11 years) and 10 in Stratum B (median age=13 years). There were no grade 4 treatment-related adverse events (TRAE). Injection site reaction was the most commonly reported TRAE. Overall survival (OS) at 12 months was 40% (95% CI, 22% to 73%) for Stratum A and 39% (95% CI, 16% to 93%) for Stratum B. The median OS was 16.1 months in patients exhibiting an expansion of H3.3K27M-reactive CD8+ T-cells compared to 9.8 months for their counterparts (p=0.05). DIPG patients with below-median baseline levels of myeloid-derived suppressor cells had prolonged OS compared to their counterparts (p<0.01). Immediate pre-treatment dexamethasone administration inversely associated with H3.3K27M-reactive CD8+ T-cell responses. Conclusion: Administration of the H3.3K27M-specific vaccine is well tolerated. Patients with H3.3K27M-specific CD8+ immunological responses demonstrated prolonged OS compared to non-responders.
Sabine Mueller, Jared M. Taitt, Javier E. Villanueva-Meyer, Erin R. Bonner, Takahide Nejo, Rishi R Lulla, Stewart Goldman, Anu Banerjee, Susan N. Chi, Nicholas S. Whipple, John R. Crawford, Karen Gauvain, Kellie J. Nazemi, Payal B. Watchmaker, Neil D. Almeida, Kaori Okada, Andres M. Salazar, Ryan D. Gilbert, Javad Nazarian, Annette M. Molinaro, Lisa H. Butterfield, Michael D. Prados, Hideho Okada