Acute graft-versus-host disease (aGvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT) inflicted by alloreactive T cells primed in secondary lymphoid organs (SLOs) and subsequent damage to aGvHD target tissues. In recent years, regulatory T cell (Treg) transfer and/or expansion has emerged as a promising therapy to modulate aGvHD. However, cellular niches essential for fostering Tregs to prevent aGvHD have not been explored, yet. Here, we tested whether and to what extent MHC class II (MHCII) expressed on Ccl19+ fibroblastic reticular cells (FRCs) shape the donor CD4+ T cell response during aGvHD. Animals lacking MHCII expression on Ccl19-Cre-expressing FRCs (MHCIIΔCcl19) showed aberrant CD4+ T cells activation in the effector phase resulting in exacerbated aGvHD that was associated with significantly reduced expansion of Foxp3+ Tregs and invariant natural killer T (iNKT) cells. Skewed Treg maintenance in MHCIIΔCcl19 mice resulted in loss of protection from aGvHD provided by adoptively transferred donor Tregs. In contrast, although FRCs upregulated co-stimulatory surface receptors, degraded and processed exogenous antigens after myeloablative irradiation, FRCs were dispensable to activate alloreactive CD4+ T cells in two mouse models of aGvHD. In sum, these data reveal an immunoprotective, MHCII-mediated function of FRC niches in secondary lymphoid organs (SLOs) after allo-HCT and highlights a hitherto unknown framework of cellular and molecular interactions that regulate CD4+ T cell alloimmunity.
Haroon Shaikh, Joern Pezoldt, Zeinab Mokhtari, Juan Gamboa Vargas, Duc-Dung Le, Josefina Peña Mosca, Estibaliz Arellano-Viera, Michael A.G. Kern, Caroline Graf, Niklas Beyersdorf, Manfred B. Lutz, Angela Riedel, Maike Büttner-Herold, Alma Zernecke, Hermann Einsele, Antoine-Emmanuel Saliba, Burkhard Ludewig, Jochen Huehn, Andreas Beilhack
Acquired aplastic anemia (AA) is caused by autoreactive T-cell-mediated destruction of early hematopoietic cells. Somatic loss of human leukocyte antigen (HLA) Class I alleles was identified as a mechanism of immune escape in surviving hematopoietic cells of some AA patients. However, pathogenicity, structural characteristics and clinical impact of specific HLA alleles in AA remain poorly understood. Here, we evaluated somatic HLA loss in 505 AA patients from two multi-institutional cohorts. Using a combination of HLA mutation frequencies, peptide-binding structures, and association with AA in an independent cohort of 6,323 patients from the National Marrow Donor Program, we identified 19 AA risk alleles and 12 non-risk alleles and established a novel AA HLA pathogenicity stratification. Our results define pathogenicity for the majority of common HLA-A/B alleles across diverse populations. Our study demonstrates that HLA alleles confer different risks of developing AA, but once AA develops, specific alleles are not associated with response to immunosuppression or trans-plant outcomes. However, higher pathogenicity alleles, particularly HLA-B*14:02, are associated with higher rates of clonal evolution in adult AA patients. Our study provides novel insights into the immune pathogenesis of AA, opening the door to future autoantigen identification and improved under-standing of clonal evolution in AA.
Timothy S. Olson, Benjamin F. Frost, Jamie L. Duke, Marian Dribus, Hongbo M. Xie, Zachary D. Prudowsky, Elissa Furutani, Jonas Gudera, Yash B. Shah, Deborah Ferriola, Amalia Dinou, Ioanna Pagkrati, Soyoung Kim, Yixi Xu, Meilun He, Shannon Zheng, Sally Nijim, Ping Lin, Chong Xu, Taizo Nakano, Joseph H. Oved, Beatriz M. Carreno, Yung-Tsi Bolon, Shahinaz M. Gadalla, Steven G.E. Marsh, Sophie Paczesny, Stephanie J. Lee, Dimitrios S. Monos, Akiko Shimamura, Alison A. Bertuch, Loren Gragert, Stephen Spellman, Daria V. Babushok
Antithrombin, a major endogenous anticoagulant, is a serine protease inhibitor (serpin). We characterized the biological and clinical impact of variants involving C-terminal antithrombin. We performed comprehensive molecular, cellular, and clinical characterization of patients with C-terminal antithrombin variants from a cohort of 444 unrelated individuals with confirmed antithrombin deficiency. We identified 17 patients carrying 12 C-terminal variants, 5 of whom had the p.Arg445Serfs*17 deletion. Five missense variants caused qualitative deficiency, and 7, including 4 insertion-deletion variants, induced severe quantitative deficiency, particularly p.Arg445Serfs*17 (antithrombin <40%). This +1 frameshift variant had a molecular size similar to that of WT antithrombin but possessed a different C-terminus. Morphologic and cotransfection experiments showed that recombinant p.Arg445Serfs*17 was retained at the endoplasmic reticulum and had a dominant-negative effect on WT antithrombin. Characterization of different 1+ frameshift, aberrant C-terminal variants revealed that protein secretion was determined by frameshift site. The introduction of Pro441 in the aberrant C-terminus, shared by 5 efficiently secreted variants, partially rescued p.Arg445Serfs*17 secretion. C-terminal antithrombin mutants have notable heterogeneity, related to variant type and localization. Aberrant C-terminal variants caused by 1+ frameshift, with similar size as WT antithrombin, may be secreted or not, depending on frameshift site. The severe clinical phenotypes of these genetic changes are consistent with their dominant-negative effects.
Carlos Bravo-Pérez, Mara Toderici, Joseph E. Chambers, José A. Martínez-Menárguez, Pedro Garrido-Rodriguez, Horacio Pérez-Sanchez, Belén de la Morena-Barrio, José Padilla, Antonia Miñano, Rosa Cifuentes-Riquelme, Vicente Vicente, Maria L. Lozano, Stefan J. Marciniak, Maria Eugenia de la Morena-Barrio, Javier Corral
POEMS syndrome is a rare monoclonal plasma cell disorder with unique symptoms distinct from other plasma cell neoplasms, including high serum VEGF levels. Since the prospective isolation of POEMS clones has not yet been successful, their real nature remains unclear. We herein performed the single-cell RNA sequencing of bone marrow plasma cells from patients with POEMS syndrome and identified POEMS clones that had immunoglobulin λ light chain (IGL) sequences (IGLV1-36, 40, 44, and 47) with amino acid changes specific to POEMS syndrome. The proportions of POEMS clones in plasma cells were markedly smaller (median: 12.9%) than in multiple myeloma (MM) (96–100%) and monoclonal gammopathy of undetermined significance (MGUS) patients (57–81%). Single-cell transcriptomes revealed that POEMS clones were CD19-negative, CD138-positive, and MHC class II-low, which allowed for their prospective isolation. POEMS clones expressed significantly lower levels of c-MYC and CCND1 than MM, accounting for their small size. VEGF mRNA was not up-regulated in POEMS clones, directly indicating that VEGF is not produced by POEMS clones. These results reveal unique features of POEMS clones and enhance our understanding of the pathogenesis of POEMS syndrome.
Yusuke Isshiki, Motohiko Oshima, Naoya Mimura, Kensuke Kayamori, Yurie Miyamoto-Nagai, Masahide Seki, Yaeko Nakajima-Takagi, Takashi Kanamori, Eisuke Iwamoto, Tomoya Muto, Shokichi Tsukamoto, Yusuke Takeda, Chikako Ohwada, Sonoko Misawa, Jun-ichiro Ikeda, Masashi Sanada, Satoshi Kuwabara, Yutaka Suzuki, Emiko Sakaida, Chiaki Nakaseko, Atsushi Iwama
Heterozygous mutations in FLT3ITD, TET2, and DNMT3A are associated with hematologic malignancies in humans. In patients, cooccurrence of mutations in FLT3ITD combined with TET2 (TF) or FLT3ITD combined with DNMT3A (DF) are frequent. However, in some rare complex acute myeloid leukemia (AML), all 3 mutations cooccur — i.e., FLT3ITD, TET2, and DNMT3A (TFD). Whether the presence of these mutations in combination result in quantitative or qualitative differences in disease manifestation has not been investigated. We generated mice expressing heterozygous Flt3ITD and concomitant for either heterozygous loss of Tet2 (TF) or Dnmt3a (DF) or both (TFD). TF and DF mice did not induce disease early on, in spite of similar changes in gene expression; during the same time frame, an aggressive form of transplantable leukemia was observed in TFD mice, which was mostly associated with quantitative but not qualitative differences in gene expression relative to TF or DF mice. The gene expression signature of TFD mice showed remarkable similarity to the human TFD gene signature at the single-cell RNA level. Importantly, TFD-driven AML responded to a combination of drugs that target Flt3ITD, inflammation, and methylation in a mouse model, as well as in a PDX model of AML bearing 3 mutations.
Baskar Ramdas, Palam Lakshmi Reddy, Raghuveer Singh Mali, Santhosh Kumar Pasupuleti, Ji Zhang, Mark R. Kelley, Sophie Paczesny, Chi Zhang, Reuben Kapur
The proteasome inhibitors (PIs) bortezomib and carfilzomib, which target proteasome 20S subunit beta 5 (PSMB5) in cells, are widely used in multiple myeloma (MM) treatment. In this study, we demonstrated the role of interferon-stimulated 20 kD exonuclease-like 2 (ISG20L2) in MM PI resistance. Gain- and loss-of-function studies showed that ISG20L2 suppressed MM cell sensitivity to PIs in vitro and in vivo. Patients with ISG20L2-low MM had a better response to PIs and a longer overall survival than patients with ISG20L2-high MM. Biotinylated-bortezomib pull-down assays showed that ISG20L2 competed with PSMB5 in binding to bortezomib. The surface plasmon resonance (SPR) assay further confirmed the direct binding of bortezomib to ISG20L2. In ISG20L2-high MM cells, ISG20L2 attenuated the binding of bortezomib to PSMB5, resulting in lower inhibition of proteasome activity and therefore less bortezomib-induced cell death. Overall, we identified a novel mechanism by which ISG20L2 conferred bortezomib resistance on MM. The expression of ISG20L2 correlated with MM PI responses and patient treatment outcomes.
Yan Yang, Yuhan Gao, Jingcao Huang, Zhuang Yang, Hongmei Luo, Fangfang Wang, Juan Xu, Yushan Cui, Hong Ding, Zhimei Lin, Xinyu Zhai, Ying Qu, Li Zhang, Ting Liu, Lingqun Ye, Ting Niu, Yuhuan Zheng
Individuals with beta-thalassemia or Sickle Cell Disease and hereditary persistence of fetal hemoglobin (HPFH) possessing 30% HbF appear to be symptom-free. Here, we used a non-integrating HDAd5/35++ vector expressing a highly efficient and accurate version of an adenine base editor (ABE8e) to install, in vivo, a -113A>G HPFH mutation in the gamma-globin promoters in “healthy” CD46/β-YAC mice carrying the human β-globin locus. Our in vivo hematopoietic stem cell (HSC) editing/selection strategy involves only subcutaneous and intravenous injections and does not require myeloablation and HSC transplantation. In vivo HSC base editing in CD46/β-YAC mice resulted in >60% -113A>G conversion with 30% γ-globin of human beta globin expressed in 70% of erythrocytes. Importantly, no off-target editing at sites predicted by CIRCLE-Seq or in silico was detected. Furthermore, no critical alterations in the transcriptome of in vivo edited mice were found by RNA-seq. In vitro, in HSCs from beta-thalassemia and Sickle Cell Disease patients, transduction with the base editor vector mediated efficient -113 A>G conversion and reactivation of γ-globin expression with subsequent phenotypic correction of erythroid cells. Because our in vivo base editing strategy is safe and technically simple, it has the potential for clinical application in developing countries where hemoglobinopathies are prevalent.
Chang Li, Aphrodite Georgakopoulou, Gregory A. Newby, Kelcee A. Everette, Evangelos Nizamis, Kiriaki Paschoudi, Efthymia Vlachaki, Sucheol Gil, Anna K. Anderson, Theodore Koob, Lishan Huang, Hongjie Wang, Hans-Peter Kiem, David R. Liu, Evangelia Yannaki, André Lieber
Increased red cell distribution width (RDW), which measures erythrocyte volume (MCV) variability (anisocytosis), has been linked to early mortality in many diseases and in older adults through unknown mechanisms. Hypoxic stress has been proposed as a potential mechanism. However, experimental models to investigate the link between increased RDW and reduced survival are lacking. Here, we show that lifelong hypobaric hypoxia (~10% O2) increases erythrocyte numbers, hemoglobin and RDW, while reducing longevity in male mice. Compound heterozygous knockout (chKO) mutations in succinate dehydrogenase (Sdh; mitochondrial complex II) genes Sdhb, Sdhc and Sdhd reduce Sdh subunit protein levels, RDW, and increase healthy lifespan compared to wild-type (WT) mice in chronic hypoxia. RDW-SD, a direct measure of MCV variability, and the standard deviation of MCV (1SD-RDW) show the most statistically significant reductions in Sdh hKO mice. Tissue metabolomic profiling of 147 common metabolites shows the largest increase in succinate with elevated succinate to fumarate and succinate to oxoglutarate (2-ketoglutarate) ratios in Sdh hKO mice. These results demonstrate that mitochondrial complex II level is an underlying determinant of both RDW and healthy lifespan in hypoxia, and suggest that therapeutic targeting of Sdh might reduce high RDW-associated clinical mortality in hypoxic diseases.
Bora E. Baysal, Abdulrahman A. Alahmari, Tori C. Rodrick, Debra Tabaczynski, Leslie Curtin, Mukund Seshadri, Drew R. Jones, Sandra Sexton
NLRP3 inflammasome and interferon stimulated gene (ISG) induction are key biological drivers of ineffective hematopoiesis and inflammation in Myelodysplastic Syndromes (MDS). Gene mutations involving messenger RNA splicing and epigenetic regulatory pathways induce inflammasome activation and myeloid lineage skewing in MDS through yet undefined mechanisms. Using immortalized murine hematopoietic stem and progenitor cells harboring these somatic gene mutations and primary MDS bone marrow specimens, we show accumulation of unresolved R-loops and micronuclei with concurrent activation of the cytosolic sensor, cGAS. cGAS-STING signaling caused interferon stimulated gene (ISG) induction, NLRP3 inflammasome activation, and maturation of the effector protease, caspase-1. Deregulation of RNA polymerase III drives cytosolic R-loop generation, which upon inhibition, extinguishes ISG and inflammasome response. Mechanistically, caspase-1 degrades the master erythroid transcription factor, GATA1, provoking anemia and myeloid lineage bias that is reversed by cGAS inhibition in vitro and in Tet2-/- hematopoietic stem and progenitor cell transplanted mice. Together, these data identity a novel mechanism by which functionally distinct mutations converge upon the cGAS-STING-NLRP3 axis in MDS directing ISG induction, pyroptosis and myeloid lineage skewing.
Amy F. McLemore, Hsin-An Hou, Benjamin S. Meyer, Nghi B. Lam, Grace A. Ward, Amy L. Aldrich, Matthew A. Rodrigues, Alexis Vedder, Ling Zhang, Eric Padron, Nicole D. Vincelette, David A. Sallman, Omar Abdel-Wahab, Alan F. List, Kathy L. McGraw
Rearrangements that drive ectopic MEF2C expression have recurrently been found in human early thymocyte progenitor acute lymphoblastic leukemia (ETP-ALL) patients. Here we show high levels of MEF2C expression in ETP-ALL patients. Using both in vivo and in vitro models of ETP-ALL, we demonstrate that elevated MEF2C expression blocks NOTCH-induced T cell differentiation while promoting a B-lineage program. MEF2C activates a B cell transcriptional program in addition to RUNX1, GATA3 and LMO2, upregulates the IL7R and boosts cell survival by upregulation of BCL2. MEF2C and the Notch pathway therefore demarcate opposite regulators of B- or T-lineage choices, respectively. Enforced MEF2C expression in mouse or human progenitor cells effectively blocks early T cell differentiation and promotes the development of bi-phenotypic lymphoid tumors that co-express CD3 and CD19, resembling human mixed phenotype acute leukemia (MPAL). SIK inhibitors impair MEF2C activity and alleviate the T cell development block. Importantly, this sensitizes cells to prednisolone treatment. Therefore, SIK inhibiting compounds such as dasatinib are potentially a valuable addition to standard chemotherapy for human ETP-ALL.
Kirsten Canté-Barrett, Mariska T. Meijer, Valentina Cordo', Rico Hagelaar, Wentao Yang, Jiyang Yu, Willem K. Smits, Marloes E. Nulle, Joris P. Jansen, Rob Pieters, Jun J. Yang, Jody J. Haigh, Steven Goossens, Jules P.P. Meijerink
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