The Journal of Experimental Medicine

Chang, Qing;Ma, Li;Lin, Ziying;Shu, Yang;Ko, Pao-Fen;Blumberg, Matthew;Chen, Jian-Fu

doi: 10.1084/jem.20241971pmid: 40970910

Whether and where lymphatic vessels occur in bone or bone marrow is unclear. The heterogeneity of skull channels and bone marrow remains poorly understood. Here, we used organ clearing, high-resolution three-dimensional imaging, cell type–specific mouse genetics, and surgical approaches to investigate skull vasculatures. We identified lymphatic vessels at the skull periosteum and found no evidence of lymphatic vessels in the cortical bones or skull bone marrow, where the lymphatic marker VEGFR3 labels blood vessels. Skull periosteum channels to the upper skin are found to occur more frequently in the parietal bone than the interparietal bone, whereas bone marrow is found more often in the interparietal bone than the parietal bone. Despite skull bone marrow expansion during aging, skull channels are significantly reduced, suggesting the aging-dependent uncoupling of skull channels and bone marrow. Together, our findings show lymphatic vessels are present in the skull periosteum but absent in bone marrow, with channel and bone marrow heterogeneity varying by skull region and age.

Han, Tao;Ruan, Chenchen;Lin, Huiyong;Zhang, Yuxia;Li, Lang;Sun, Ye-hsuan;Zhong, Chuan-Qi;Chen, Xin;Huang, Kai;Cao, Yating;Fan, Zusen;Zhang, Hongbing;Han, Jiahuai;Zhang, Yingying

doi: 10.1084/jem.20250277pmid: 40996440

Excess TNF causes systemic inflammatory response syndrome and mortality. RIPK1 coordinates TNF signaling through kinase-dependent and -independent mechanisms. S161 autophosphorylation is a primary function of RIPK1 kinase activity in vitro, and here we show that it is sufficient to mediate RIPK1 kinase–dependent function in vivo. S161 phospho-mimic mutation (S161E) effectively overcomes chemical or genetic inhibition of RIPK1 kinase activity in TNF-treated cells and mice. Mechanistically, S161 autophosphorylation is necessary for further autophosphorylation in RIPK1, including at S166. Ripk1S161E/S161E mice are hypersensitive to TNF, enabling us to observe low-dose TNF-induced necroptosis in cecal intestinal epithelial cells (IECs) and endothelial cells (ECs) and uncover a reciprocal enhancement between IEC and EC necroptosis and a selective increase of IL-6 in the circulation by necroptosis. IL-6 promotes cecal edema and synergizes with IEC and EC necroptosis, causing cecal damage and mouse death. Our data elucidate a mechanism of RIPK1 kinase–dependent function in TNF signaling and its role in cecal pathology and mouse mortality.

Hatterschide, Joshua;Yang, Liheng;Coyne, Carolyn B.

doi: 10.1084/jem.20250448pmid: 40965510

The placenta combats mother-to-fetus transmission of viruses through the antiviral activities of fetal-derived trophoblasts. Placental trophoblasts employ specialized antiviral strategies to protect against infection while preventing maternal immune rejection of the fetus. However, the full extent of how trophoblasts respond to viral infections is not well understood. To address this, we defined the transcriptional landscape of human trophoblast organoids infected with seven diverse teratogenic viruses. We found that herpesviruses, including HSV-1, HSV-2, and HCMV, did not trigger an IFN response. Instead, they activated the expression of DUX4 and its downstream target genes: DUX4-stimulated genes (DSGs). This program was enriched in trophoblasts and associated with cells containing low HSV-1 gene expression following infection. Screening highly expressed DSGs revealed that many of them exhibited anti-herpesvirus activity, indicating they comprise an alternative antiviral pathway similar to the IFN-stimulated gene response. These findings identify DUX4 as a master regulator of an antiviral program in trophoblasts, specifically targeting a prominent family of teratogenic viruses.

Stetsenko, Volodymyr;Gail, Daniel P.;Reba, Scott M.;Suzart, Vinicius G.;Schauner, Robert;Sandhu, Avinaash K.;Sette, Alessandro;Haj Dezfulian, Mohammad;Lindestam Arlehamn, Cecilia S.;Carpenter, Stephen M.

doi: 10.1084/jem.20250460pmid: 40990918

CD4+ T cell–mediated control of tuberculosis (TB) requires recognition of macrophages infected with Mycobacterium tuberculosis (Mtb). Yet, not all Mtb-specific T cells recognize infected macrophages. Using infected monocyte-derived macrophages and autologous memory CD4+ T cells from individuals with stable latent Mtb infection (LTBI), we quantify the frequency of activated T cells. T cell antigen receptor (TCR) sequencing revealed >70% of unique and >90% of total Mtb-specific TCR clonotypes in LTBI are linked to recognition of infected macrophages, while a subset required exogenous antigen exposure, suggesting incomplete recognition. Clonotypes specific for multiple Mtb antigens, and other pathogens, were identified. Remarkably, antigen screening revealed all TCRs to be specific for type VII secretion system (T7SS) substrates. Mtb-specific clonotypes expressed signature effector functions dominated by IFNγ, TNF, IL-2, and GM-CSF or chemokine production and signaling. We propose that TB vaccines, which elicit T cells specific for T7SS substrates, recognize infected macrophages, and express canonical effector functions, will offer protection against TB.

Koerner, Lioba;Li, Xiaoming;Silnov, Eveline;Laurien, Lucie;Pasparakis, Manolis

doi: 10.1084/jem.20250279pmid: 40996439

RIPK1 regulates cell death and inflammation and has been implicated in the pathogenesis of inflammatory diseases. RIPK1 autophosphorylation promotes cell death induction; however, the underlying mechanisms and the role of specific autophosphorylation sites remain elusive. Using knock-in mouse models, here we show that S161 autophosphorylation has a critical physiological function in RIPK1-mediated cell death and inflammation. S161N substitution partially suppressed RIPK1-mediated catalytic activity and cell death induction but was sufficient to prevent skin inflammation induced by keratinocyte necroptosis or apoptosis in relevant mouse models. Combined S161N and S166A mutations synergized to prevent RIPK1-mediated cell death more efficiently than the single site mutations, revealing functional redundancy. Moreover, phosphomimetic S161E mutation could overcome the necroptosis-inhibitory effect of S166A mutation, revealing that S161 phosphorylation is sufficient for necroptosis induction. Collectively, a functional interplay of S161 and S166 phosphorylation events regulates RIPK1-dependent cell death and inflammation.

Ishikawa, Eri;Kosako, Hidetaka;Motooka, Daisuke;Imasaka, Mai;Watarai, Hiroshi;Ohmuraya, Masaki;Yamasaki, Sho

doi: 10.1084/jem.20250541pmid: 40965511

Development of invariant natural killer T (iNKT) cells in the thymus requires cell–cell interaction through invariant TCR (iTCR) and CD1d, which induces expression of the transcription factor, promyelocytic leukemia zinc finger (PLZF). However, the signaling pathway linking iTCR and PLZF remains unclear. Here, we report that a serine/threonine kinase, protein kinase D (PKD), plays a pivotal role in iNKT cell development. In T cell–specific PKD-deficient (Prkd2/3∆CD4) mice, PLZF induction and iNKT cell generation were severely impaired, which were rescued by introduction of a PLZF transgene. We identified the transcription factor Ikaros as a substrate of PKD upon iTCR stimulation. Knock-in mice carrying a phosphorylation-defective mutant Ikaros (Ikzf1S267/275A) exhibited an impairment of iNKT cell development, whereas conventional T cells were normal. In iNKT cells, Ikaros binds to the upstream region of the PLZF gene to induce its transcription. Mutant mice lacking the Ikaros-binding site (Zbtb16∆IBS) generated fewer iNKT cells than WT mice. These results suggest that PKD links iTCRs to PLZF induction through Ikaros, thereby mediating iNKT cell development.

Branchett, William J.;Stavropoulos, Evangelos;Shields, Jessica;Al-Dibouni, Alaa;Cardoso, Marcos;Fernandes, Ana Isabel;Moreira-Teixeira, Lúcia;Slawinski, Hubert;Mikolajczak, Anna;Rodgers, Angela;Saraiva, Margarida;O’Garra, Anne

doi: 10.1084/jem.20250466pmid: 40986319

The early immune mechanisms determining Mycobacterium tuberculosis infection outcome are unclear. Using bulk and scRNA-seq over the first weeks of infection, we describe an unexpected, higher early pulmonary type I IFN response in relatively resistant C57BL/6 as compared with highly TB-susceptible C3HeB/FeJ mice. C57BL/6 mice showed pronounced early monocyte-derived macrophage (MDM) accumulation and extensive CD4+ T cell–MDM interactions in lung lesions, accompanied by high expression of T cell–attractant chemokines by MDMs. Conversely, lesions in C3HeB/FeJ mice were dominated by neutrophils with high expression of pro-inflammatory chemokines, from which CD4+ T cells were spatially segregated. Early type I IFN signaling blockade reduced bacterial load and neutrophil swarming within early TB lesions while increasing CD4+ T cell numbers in both C57BL/6 and C3HeB/FeJ mice, with later more pronounced effects on bacterial load in C3HeB/FeJ mice. These data suggest that early type I IFN signaling during M. tuberculosis infection favors neutrophil accumulation and limits CD4+ T cell infiltration into developing lesions.

Florescu, Alexandra;Zuo, Michelle;Wang, Angela A.;Champagne-Jorgensen, Kevin;Noor, Mohammed A.;Ward, Lesley A.; van Puijenbroek, Erwin;Klein, Christian;Gommerman, Jennifer L.

doi: 10.1084/jem.20241255pmid: 40965509

In multiple sclerosis (MS), the leptomeninges (LM) are populated with immune cell aggregates that correlate with disease progression. The impact of LM inflammation on the adjacent dura is largely unknown. Using a mouse model of MS that induces brain LM inflammation and age-dependent disease progression, we found that encephalitogenic T cells and B220high B cells accumulate substantially in the brain LM and parenchyma of both young and aged mice, while the adjacent dura remains relatively inert. We also observed a population of anti-CD20–resistant B220low B cells in the dura and bone marrow that virtually disappear at disease onset and accumulate in the brain of young mice concomitant with disease remission. In contrast, aged mice show a paucity of brain-resident B220low B cells at the expense of class-switched B220high B cells accompanied by severe, chronic disease. In summary, dynamic changes in the brain, LM, and dural B cells are associated with age-dependent disease severity in an animal model of progressive MS.

Zhu, Kaiwen;Dou, Jiayu;Li, Bin;Qian, Jin;Zhong, Ming-Chao;Tang, Zhenghai;Lu, Yan;Veillette, André

doi: 10.1084/jem.20250431pmid: 41026163

MAIT cells are innate-like T cells known for their semi-invariant TCR that recognizes vitamin B metabolites presented by MR1. While the involvement of TCR and cytokines in development and activation of MAIT cells is well documented, the contribution of co-receptors, including SLAM family receptors, remains poorly understood. This study revealed that CD2 and its ligands, CD48 in mice and CD58 in humans, were crucial for MAIT cell maturation and antigen-driven activation, but not for their responses to cytokines. Cis interactions of CD2 with its ligands on the same cell were essential for activation, with trans interactions contributing in some contexts. A natural subset of human MAIT cells lacking CD2 displayed reduced activation responses to antigen. Human CD48 recognized 2B4 rather than CD2, dampening TCR signal strength and activation of human MAIT cells. Thus, the interplay between CD2 and its ligands is pivotal for MAIT cell development and activation, highlighting potential approaches for treating human diseases implicating MAIT cells.