Immunology
Abstract
The human retina and underlying choroid have multiple well-organized vascular layers. However, retinal and choroidal neovascularization can develop in diseases such as diabetic retinopathy or neovascular age-related macular degeneration, which can result in destructive vision loss. Ocular macrophages are implicated in the pathogenesis of such neovascular diseases. To identify a potential pro-angiogenic macrophage subtype, we integrated 13 single-cell RNA sequencing datasets from human and mouse eyes comprising 173 unique samples. From 99,184 mononuclear phagocytes, we identified a single macrophage subtype with high expression of SPP1, FABP5, and IL7R that was over-represented from neovascular disease samples. We fate mapped these cells and specifically depleted them, showing that they are monocyte-derived and necessary for laser-induced choroidal neovascularization in mice. Finally, we performed a high-resolution spatial RNA sequencing experiment on six human donors and found that the pro-angiogenic macrophage subtype localized to human choroidal neovascular membranes. These macrophages and other cells in neovascular membranes expressed MMP14, a potential therapeutic target, and MMP14 inhibition decreased choroidal neovascularization in mice. Collectively, we show that this Monocyte-derived Pro-angiogenic Ocular Macrophage (MoPOM) subtype is upregulated in multiple ocular neovascular diseases across species, necessary for choroidal neovascularization, and is a potential therapeutic target to reduce destructive angiogenesis in the eye.
Authors
Andrew P. Voigt, Joyce Gong, Steven Droho, Kelly Mulfaul, Amrita Rajesh, Greta E. Forbes, Jacob K. Sterling, Miles J. Flamme-Wiese, Robert F. Mullins, Jeremy A. Lavine
Abstract
Splenic myelopoiesis supplies immunosuppressive myeloid cells in cancer, yet its local regulation remains unclear. Here we identify a self-amplifying circuit between neutrophils and hematopoietic stem and progenitor cells (HSPCs) that sustains splenic myelopoiesis. Tumor-associated neutrophils produced reactive oxygen species (ROS) and created an oxidatively stressed milieu in the spleen, which activated FOXO1 in neighboring HSPCs and drove DRP1-mediated mitochondrial fission, reprogramming HSPCs toward myeloid-biased expansion and immunosuppressive output. Progeny neutrophils reinforced splenic oxidative stress and perpetuated this loop. Genetic ablation of Cybb or spleen-targeted ROS scavenging inhibited FOXO1 activation and preserved fused mitochondria morphology in HSPCs. Consistently, blockade of FOXO1 or DRP1 prevented mitochondrial fission and reduced ROS-producing myeloid cell generation. Such interventions reduced splenic myelopoiesis, curtailed the production of suppressive myeloid cells in the spleen and their infiltration into tumors, and enhanced cytotoxic T cell activity, thereby restoring antitumor immunity and restraining tumor progression. Analyses of spleen samples from cancer patients, together with cord-blood HSPC and neutrophil co-culture experiments, support the human relevance of this neutrophil–ROS–HSPC axis. These findings reveal a self-propagating neutrophil–HSPC feedback circuit that locally sustains tumor-promoting splenic myelopoiesis and provide a rationale for targeting neutrophil-to-progenitor signaling to normalize antitumor immunity.
Authors
Lan Zhu, Shufeng Luo, Qiaomin Hua, Chang-An Zhao, Huiling Lin, Mingyu Liu, Lingyan Zhu, Jiabin Zheng, Huolun Feng, Yong Li, Chong Wu, Limin Zheng
Abstract
Hypoxia-inducible factor 2α (HIF-2α) is a central oncogenic driver in clear cell renal cell carcinoma (ccRCC) and a therapeutic target of the small-molecule inhibitor belzutifan. Genetic studies in murine models have suggested that hypoxia signaling may support T cell effector programs, raising concern that HIF-2α inhibition could impair antitumor immunity. However, whether pharmacologic HIF-2α inhibition alters human T cell biology remains unknown. Here, we investigated the cell-intrinsic effects of EPAS1 (encoding HIF-2α) perturbation in primary human T cells. CRISPR/Cas9-mediated deletion of HIF-2α demonstrated no notable transcriptional effects. Similarly, pharmacologic treatment with belzutifan produced minimal transcriptional changes and did not impair proliferation, cytokine production, polyfunctionality, or cytotoxic activity in T cells derived from healthy donor peripheral blood, peripheral blood from patients with ccRCC, or tumor-infiltrating lymphocytes under hypoxic conditions. High- dimensional immunophenotyping revealed preserved T cell differentiation and activation states following pharmacologic HIF-2α inhibition in vitro and in peripheral blood from patients receiving HIF-2α inhibitor therapy. Together, these findings demonstrate that pharmacologic HIF-2α inhibition preserves key effector programs, providing a mechanistic basis for the immunologic safety of HIF-2α–targeted therapy in ccRCC.
Authors
Katrine N. Madsen, Soki Kashima, Hanna Soulati, Lena Wirth, Katsuhiro Ito, Zachary A. Yochum, Vivien Moritz, Julia Walker, Marc Machaalani, Fady Ghali, Patrick Kenney, Adebowale Adeniran, Michael Hurwitz, Toni K. Choueiri, David A. Braun
Receptor-tuned Interleukin-2 Mutein Decouples Treg Expansion from ILC2-driven Cutaneous Inflammation
Abstract
Low-dose interleukin-2 (IL-2) and IL-2 muteins are being developed to expand regulatory T cells (Tregs) for autoimmune disease therapy, but injection site reactions (ISRs) remain frequent and poorly understood. Here we show IL2Mut24, a murine surrogate of IL-2 receptor α (IL-2Rα, CD25)-biased IL-2 mutein, enhances Treg expansion yet paradoxically exacerbates cutaneous inflammation compared to wild-type IL-2. Using immunodeficient mice, antibody blockade and adoptive transfer, we identify group 2 innate lymphoid cells (ILC2s) as key drivers of IL-2-induced skin inflammation, defining a CD25-dependent innate activation axis that constrains immune tolerance. In cynomolgus monkeys, the CD25-biased IL-2 mutein efavaleukin alfa promotes greater peripheral Treg expansion than aldesleukin (recombinant human IL-2) but is accompanied by transient increases in IL-5 and dose-dependent ISRs, indicating conservation of this innate inflammatory program across species. To improve the therapeutic window, we engineer receptor clamps by linking IL2Mut24 to antibodies against CD25 to restrict IL-2 access to CD25. This receptor-tuned IL-2 preserves Treg selectivity, suppresses ISRs, and outperforms IL2Mut24 in experimental autoimmune encephalomyelitis by restraining Th17 responses. These findings reveal a conserved innate mechanism underlying IL-2–associated toxicity and establish receptor-tuning as a strategy to improve the safety and efficacy of IL-2–based immunotherapy.
Authors
Anupama Sahoo, Cody Moorman, Mina Tsenkova, Yi Jing, Alexis Valdovinos, Xin Luo, Shiping Lu, Songyu Wang, Renee R. Hukkanen, Madeline Fort, Helen S.H. Tang, Ronya Primack, Shweta Mandavalli, Weiwen Deng
Abstract
Authors
Tony Yao, Rita E. Chen, Melissa Yamada, J. Russell Moore, Marissa Jimenez, Tammy Huang, Lynn Cornelius, George Ansstas, Naresha Saligrama, David Y. Chen
Abstract
Receptor activator of NFκB ligand (RANKL) is important for bone metabolism, but also modulates immune processes. We showed that mast cells (MCs) are involved in RANKL regulation, but the importance of MC-derived RANKL in skin inflammation has not yet been investigated. In contact hypersensitivity (CHS), the absence of MC-derived RANKL led to reduced skin inflammation due to impaired leukocyte infiltration and blood lymphopenia. Surprisingly, we observed a massive hyperplasia of the distant inguinal lymph nodes in the absence of MC-RANKL. Using adoptive transfers, flow cytometry and whole-mount 3D imaging, we demonstrated that this was not caused by structural maladaptation, but rather by the inability of lymphocytes to exit in a timely manner. Importantly, RANKL deletion in skin MCs only replicated the effect of LN hyperplasia and blood lymphopenia. Moreover, MCs were involved in serum sphingosine-1-phosphate (S1P) regulation during sensitization and challenge. Intravascular administration of S1P restored timely lymphocyte egress, demonstrating a MC-induced organ-spanning RANKL-S1P axis. Consequently, peripheral skin MC-derived RANKL is essential for the timely lymphocyte egress from distant LNs, which may have important implications for the targeted treatment of inflammatory skin diseases.
Authors
Konstantinos Katsoulis-Dimitriou, Waqar Umer, Laura Knop, Tanja Schickschneit, Aaron Hoffmann, Ali El-Bizri, Lea M. Schmitter, Kathleen Baumgart, Nouria Jantz-Naeem, Vladyslava Dovhan, Charlotte Heidelbach, Lars Philipsen, Andreas J. Müller, Stephan Fricke, Sascha Kahlfuß, Thomas Schüler, Jan Dudeck, Anne Dudeck
Abstract
Chronic Pseudomonas aeruginosa infection is a central driver of bronchiectasis and contributes to progressive lung decline in patients with cystic fibrosis (CF), even in the era of CFTR modulators. A major limitation in the field is that conventional mouse models fail to develop persistent, biofilm-associated lung infection, restricting mechanistic studies and preclinical evaluation. Here, we establish clinically relevant infection models by combining CF-like mouse strains (βENaC-overexpressing and CFTR-deficient mice) with agarose bead–embedded P. aeruginosa that forms persistent, tobramycin-refractory biofilms. Using intravital lung microscopy, we show that alveolar macrophages initially respond by surrounding biofilms but progressively dissociate from the biofilms during persistent infection. Neutrophils are also recruited but fail to clear bacteria. Administration of gremubamab (MEDI3902), a bispecific antibody targeting the virulence factors Psl and PcrV, preserves alveolar macrophages in persistent biofilm infection, restores bacterial sensing and phagocytosis, limits excessive neutrophilic inflammation, and significantly improves bacterial clearance and survival. Together, these findings establish a clinically relevant persistent P. aeruginosa infection model and highlight gremubamab as a promising virulence-targeted therapy to potentially overcome bacterial immune evasion while restoring host defense in mouse models of bronchiectasis and CF.
Authors
Wanhai Qin, Wayne Brailsford, Stacey M. Cromer Berman, Christina S Thornton, Antonio DiGiandomenico, Paul Kubes
Abstract
Background. Improved understanding of the cellular mechanisms of organ transplant rejection is needed for developing targeted therapies to improve outcomes. We used digital cytometry to estimate the digital leukocyte entities (DLE) shared by rejection in biopsies of kidney, heart, lung, and liver organ transplants. Methods. We used CIBERSORTx with the LM22 leukocyte reference to estimate associations between 22 DLEs with rejection in 8422 transplant biopsies - 4898 kidney, 2361 heart, 652 lung, and 538 liver. Results. In all organs, rejection correlated with an increase in DLEs designated as classically activated macrophages and a decrease in DLEs designated regulatory T cells. T cell-mediated rejection (TCMR) was associated with a massive increase in otherwise rare T cell DLEs designated memory-activated CD4 and Tγδ, as well as follicular-helper CD4 and CD8. TCMR also had increased DLE designated plasma cells, compatible with a role for the B cell lineage in antigen presentation in TCMR. Antibody-mediated rejection (ABMR) in kidney and heart transplant biopsies correlated with increased DLEs representing activated mast cells, activated NK cells, and monocytes. Some findings were validated by single-cell/nucleus RNAseq data from kidney and liver biopsies. Conclusion. The shared correlations of DLEs with rejection in all organs - e.g. CD4ma with TCMR and activated mast cells with ABMR - suggest novel aspects of rejection. These findings can guide the search for the in vivo leukocytes that correspond with the digital entities, with potential clinical applications. Trial registration. ClinicalTrials.gov NCT01299168, NCT02670408, NCT02812290, NCT03193151. Funding. Genome Canada and a grant from Natera, Inc.
Authors
Patrick T. Gauthier, Louisa M.S. Gerhardt, Lorenz Jahn, Christian Hinze, Philip F. Halloran
Abstract
Pharmacologic immunosuppression is essential for preventing organ rejection and controlling autoimmunity, but profoundly impairs humoral immunity, increasing the risk of vaccine failure and infection. The mechanisms by which immunosuppressive therapies disrupt human B cell responses remain poorly defined. Here, we identified dysregulated lipid metabolism as a central determinant of impaired vaccine response in solid organ transplant recipients (SOTRs). Using high-dimensional immune profiling, single-cell transcriptomics, and functional metabolic assays, we found that effective B cell responses required a homeostatic balance between lipid synthesis and fatty acid oxidation. The widely used immunosuppressive agent, mycophenolic acid (MPA) was strongly associated with vaccine non-response and induced excessive lipid synthesis, lipid accumulation, and mitochondrial stress in B cells. In contrast, CD11c+ B cells retained the capacity to differentiate into plasmablasts in the presence of MPA through elevated expression of CPT1A, a mitochondrial fatty acid transporter, and enhanced fatty acid oxidation. These cells were found to be a key feature of early effective vaccine responses in healthy individuals and SOTRs. Notably, pharmacologic inhibition of cholesterol synthesis partially restored plasmablast differentiation in the presence of MPA. These findings identify B cell lipid metabolism as a critical and targetable regulator of human humoral immunity during immunosuppression.
Authors
Elizabeth A. Thompson, Alexis Figueroa, Katerina Roznik, Nicole E. Skinner, Santosh Dhakal, Shuai Li, Luca Biavati, Laura A. Sena, Laila Stoddart, Karli Redinger, Samuel B. Warner, Sabra L. Klein, Nadine Rouphael, Joel N. Blankson, Yolanda Eby, Robert D. Leone, Peter S. Heeger, Mark A. Robien, Christian P. Larsen, Erika L. Pearce, Edward J. Pearce, Hongkai Ji, Andrew H. Karaba, Dorry L. Segev, Aaron A.R. Tobian, William A. Werbel, Andrea L. Cox, Justin R. Bailey
Abstract
Loss-of-function mutation in the human gene dipeptidyl peptidase 9 (DPP9) causes Hatipoglu syndrome leading to severe inflammasomopathy. A key feature of the disease is pancytopenia, and patients require bone marrow transplantation, but the mechanism of cell loss is unclear, since Dpp9-mutant mice have normal hematopoiesis, suggesting that a distinct mechanism of disease occurs in humans. Here, we present a model of human DPP9 deficiency leveraging reverse genetics in the MISTRG6 humanized mice. We found that CRISPR editing of human CD34+ hematopoietic stem and progenitor cells (HSPCs) led to very efficient and persistent gene deletion in vivo. Human DPP9 deletion recapitulated cytopenia in peripheral blood and in the bone marrow, and cell loss was cell intrinsic. However, DPP9 deletion led to few transcriptional changes suggesting posttranscriptional regulation in human HSPCs. Mechanistically, DPP9 deficiency led to activation of the CARD8 inflammasome, resulting in HSPC pyroptosis, whereas NLRP1 was dispensable for cell death. Thus, our results reveal a unique human mechanism of disease and offer therapeutic insight for this inflammasomopathy.
Authors
Tianli Xiao, J. Richard Brewer, Maximillian Carlino, Ailin Han, Yamato J. Takabe, Chia-Yi Lee, Fengrui Zhang, Mi Chen, Holly Nicole Blackburn, Amin H. Nassar, Qiankun Wang, Kristen Brennand, Liang Shan, Esen Sefik, Diane S. Krause, Richard A. Flavell
Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)