Zhaoqi Zhang

Appropriate T-cell functional polarization is critical for maintaining immune stability and immune tolerance. The role of Fam234a in the functional polarization of T cells is unknown. In a DSS-induced inflammatory bowel disease model in Rag2^-/- mice with either naive WT or Fam234a-deficient CD4⁺ T cells, mice with Fam234a-deficient CD4⁺ T cells presented milder symptoms of colitis, accompanied by a decreased ratio of Th17/Treg cells. Consistent with the in vivo observations, Th17 differentiation was significantly decreased and Treg induction was increased in the in vitro naive Fam234a-deficient CD4⁺ T-cell polarizing induction system. Similarly, knocking down FAM234A in human T cells using siRNA also revealed that FAM234A deficiency significantly decreased the Th17/Treg cell ratio in human T cells. Coimmunoprecipitation-mass spectrometry (Co-IP-MS), protein interaction, and biochemical studies revealed that FAM234A may directly interact with the deubiquitinase USP4 to affect its deubiquitination function. The reduction in Th17 cells and increase in Treg cells among Fam234a-deficient T cells were significantly reversed by restoring USP4 overexpression. RNA sequencing and molecular studies indicated that Fam234a knockout reduced USP4-mediated Rheb and RORγt deubiquitination, mTOR activation, and Hif1α expression and ultimately affected Th17 and Treg differentiation. Therefore, Fam234a intrinsically balances the Th17 and Treg differentiation of naive CD4⁺ T cells by directly preventing USP4-mediated deubiquitination of Rheb to regulate mTOR-HIF1α-related oxidative phosphorylation and glycolytic gluconeogenesis metabolism pathways as well as USP4-mediated deubiquitination of RORγt pathways. This research revealed the critical role of FAM234A in the orchestration of Th17/Treg cell fate decisions and may offer potential therapies for their related diseases.

Tissue-resident macrophages (RTMs), predominantly originating from embryonic progenitors, play a pivotal role in maintaining tissue homeostasis and immune regulation. Their functional diversity is shaped by both their developmental origins—including the yolk sac, fetal liver, and bone marrow—and the local tissue microenvironment. In this study, we identified a unique subset of RTMs in Lyz2cre-RosaYFP reporter mice that lack Lyz2 expression and persist long-term in peripheral tissues. These Lyz2-YFP- RTMs follow a distinct developmental trajectory, transitioning from Lyz2-YFP-CD117+CD45-AA4.1- early erythro-myeloid progenitors (EMPs) to Lyz2-YFP-CD117+CD45+AA4.1- intermediate EMPs in a Csf2rb-dependent manner. Transcriptional profiling reveals that yolk sac-derived Kupffer cells (KCs) lacking Lyz2 expression are enriched in genes associated with T cell chemotaxis and tumor immunity. Functionally, these Lyz2-never expressed (Lyz2-YFP-) KCs are preferentially localized within intratumoral regions, where they promote hepatocellular carcinoma progression by recruiting CD4+CD25+Foxp3+ regulatory T cells (Tregs) and inducing T cell exhaustion. Conversely, Lyz2-ever expressed (Lyz2-YFP+) KCs exhibit anti-tumor activity by enhancing effector CD8+ T cell function. Our findings unveil a long-lived subset of RTMs with sustained pro-tumorigenic potential, underscoring the critical role of their yolk sac-derived developmental trajectory in shaping their functional properties. This study provides novel insights into the ontogeny-dependent heterogeneity of RTMs and their dual roles in tumor immunity.

Pigs are increasingly recognized as promising candidates for clinical xenotransplantation and as large‐animal models for biomedical research; however, interspecies differences in gut microbiota, immune function, and metabolism remain major barriers. To address this, we established gut microbiota‐humanized (GMH) pigs by transplanting human fecal microbiota into antibiotic‐treated pigs. We systemically evaluated alterations in microbiota composition, serum metabolites, and immune cell profiles using integrated metagenomic, quasi‐targeted metabolomic and single‐cell transcriptomic (scRNA‐seq) analyses. Metagenomic profiling revealed a shift in the intestinal microbiota of GMH pigs toward a human‐like composition, characterized by enrichment of Bacteroidia and depletion of Bacilli . Metabolomic analysis showed that GMH pigs exhibited serum metabolite profiles more closely resembling those of humans. Among 423 detected serum metabolites, 136 that were lower in control pigs than in humans were upregulated in GMH pigs, whereas 79 that were elevated in control pigs decreased post‐transplantation. Notably, pathways related to tryptophan metabolism, bile acid biosynthesis, and fatty acid metabolism were enhanced in GMT pigs, while carbon‐related and glycolytic pathways were attenuated, indicating partial convergence toward human metabolic phenotype. Integration of microbial and metabolite data identified 20 and 33 metabolites associated with Bacteroidia and Bacilli , respectively. scRNA‐seq profiling of peripheral blood mononuclear cells demonstrated transcriptional and compositional remodeling of T cells, monocytes, and B cell subsets in GMH pigs. These findings demonstrated that human fecal microbiota can reshape both systemic metabolic and immune artitecture in pigs, offering a robust large‐animal platform for studying host‐microbiota interactions and advancing translational application in xenotransplantation and microbiome‐based therapeutics.

T lymphocytes are pivotal in adaptive immunity. The role of the trafficking protein particle complex (TRAPPC) in regulating T‐cell development and homeostasis is unknown. Using CD4^cre‐Trappc1^flox/flox (Trappc1 cKO) mice, we found that Trappc1 deficiency in T cells significantly decreased cell number of naive T cells in the periphery, whereas thymic T‐cell development in Trappc1 cKO mice was identical as WT mice. In the culture assays and mouse models with adoptive transfer of the sorted WT (CD45.1⁺CD45.2⁺) and Trappc1 cKO naive T cells (CD45.2⁺) to CD45.1⁺ syngeneic mice, Trappc1‐deficient naive T cells showed significantly reduced survival ability compared with WT cells. RNA‐seq and molecular studies showed that Trappc1 deficiency in naive T cells reduced protein transport from the endoplasmic reticulum to the Golgi apparatus, enhanced unfolded protein responses, increased P53 transcription, intracellular Ca²⁺, Atf4‐CHOP, oxidative phosphorylation, and lipid peroxide accumulation, and subsequently led to ferroptosis. Trappc1 deficiency in naive T cells increased ferroptosis‐related damage‐associated molecular pattern molecules like high mobility group box 1 or lipid oxidation products like prostaglandin E2, leukotriene B4, leukotriene C4, and leukotriene D4. Functionally, the culture supernatant of Trappc1 cKO naive T cells significantly promoted neutrophils to express inflammatory cytokines like TNFα and IL‐6, which was rescued by lipid peroxidation inhibitor Acetylcysteine. Importantly, Trappc1 cKO mice spontaneously developed severe autoinflammatory disease 4 weeks after birth. Thus, intrinsic expression of Trappc1 in naive T cells plays an integral role in maintaining T‐cell homeostasis to avoid proinflammatory naive T‐cell death‐caused autoinflammatory syndrome in mice. This study highlights the importance of the TRAPPC in T‐cell biology.

Fatty acid binding protein 5 (FABP5) is mainly involved in the uptake, transport, and metabolism of fatty acid in the cytoplasm, and its role in immune cells has been recognized in recent years. However, the role of FABP5 in macrophage inflammation and its underlying mechanisms were not fully addressed. In our study, the acute liver injury and sepsis mouse models were induced by i.p. injection of LPS and cecal contents, respectively. Oleic acid (0.6 g/kg) was injected four times by intragastric administration every week, and this lasted for 1 wk before the LPS or cecal content challenge. We found that myeloid-specific deletion of FABP5 mitigated LPS-induced acute liver injury with reduced mortality of mice, histological liver damage, alanine aminotransferase, and proinflammatory factor levels. Metabolic analysis showed that FABP5 deletion increased the intracellular unsaturated fatty acids, especially oleic acid, in LPS-induced macrophages. The addition of oleic acid also decreased LPS-stimulated macrophage inflammation in vitro and reduced acute liver injury in LPS-induced or cecal content–induced sepsis mice. RNA-sequencing and molecular mechanism studies showed that FABP5 deletion or oleic acid supplementation increased the AMP/ATP ratio and AMP-activated protein kinase (AMPK) activation and inhibited the NF-κB pathway during the inflammatory response to LPS stimulation of macrophages. Inhibiting AMPK activation or expression by chemical or genetic approaches significantly rescued the decreased NF-κB signaling pathway and inflammatory response in LPS-treated FABP5-knockout macrophages. Our present study indicated that inhibiting FABP5 or supplementation of oleic acid might be used for the treatment of sepsis-caused acute liver injury.

Myeloid cell development in bone marrow is essential for the maintenance of peripheral immune homeostasis. However, the role of intracellular protein trafficking pathways during myeloid cell differentiation is currently unknown. By mining bioinformatics data, we identify trafficking protein particle complex subunit 1 (TRAPPC1) as continuously upregulated during myeloid cell development. Using inducible ER-TRAPPC1 knockout mice and bone marrow chimeric mouse models, we demonstrate that TRAPPC1 deficiency causes severe monocyte and neutrophil defects, accompanied by a selective decrease in common myeloid progenitors (CMPs) and subsequent cell subsets in bone marrow. TRAPPC1-deleted CMPs differentiate poorly into monocytes and neutrophils in vivo and in vitro, in addition to exhibiting enhanced endoplasmic reticulum stress and apoptosis via a Ca²⁺ -mitochondria-dependent pathway. Cell cycle arrest and senescence of TRAPPC1-deleted CMPs are mediated by the activation of pancreatic endoplasmic reticulum kinase and the upregulation of cyclin-dependent kinase inhibitor p21. This study reveals the essential role of TRAPPC1 in the maintenance and differentiation of CMPs and highlights the significance of protein processing and trafficking processes in myeloid cell development.

Whether induced tissue-resident memory T (T _RM ) cells in nonlymphoid organs alone can mediate allograft rejection is unknown. By grafting alloskin or heart into severe combined immunodeficient or Rag2KO mice in which a piece of induced CD4 ⁺ and/or CD8 ⁺ T _RM cell–containing MHC-matched or syngeneic skin was transplanted in advance, we addressed this issue. The induced CD4 ⁺ T _RM cells in the skin alone acutely rejected alloskin or heart grafts. RNA-seq analysis showed that induced CD4 ⁺ T _RM cells in skin favorably differentiated into T _H 17-like polarization during the secondary immune response. Inhibition of the key T _H 17 signaling molecule RORγt attenuated T _RM cell–mediated graft rejection. Thus, we offer a unique mouse model to specifically study T _RM cell–mediated allograft rejection without the involvement of lymphocytes in lymphoid organs and tissues. Our study provides strong evidence supporting the hypothesis that long-lived alloreactive T _RM cells resident in other organs/tissues substantially contribute to organ allograft rejection.

Thymic epithelial cells (TECs) are critical for the development and generation of functionally competent T cells. Until now, the mechanism that regulates the survival of TECs is poorly understood. In the current study, we found that Tsc1 controls the homeostasis of medullary TECs (mTECs) by inhibiting lysosomal-mediated apoptosis pathway in mice. TEC-specific deletion of Tsc1 predominately decreased the cell number of mTECs and, to a lesser content, affected the development cortical TECs. The defect of mTECs caused by Tsc1 deficiency in mice impaired thymocyte development and peripheral T cell homeostasis. Mechanistically, Tsc1 deficiency did not affect the cell proliferation of mTECs but increased the apoptosis of mTECs significantly. RNA-sequencing analysis showed that pathways involved in lysosomal biogenesis, cell metabolism, and apoptosis were remarkably elevated in Tsc1-deficient mTECs compared with their wild-type counterparts. Tsc1-deficient mTECs exhibited overproduction of reactive oxygen species and malfunction of lysosome, with lysosome membrane permeabilization and the release of cathepsin B and cathepsin L to the cytosol, which then lead to Bid cleaved into active truncated Bid and subsequently intrinsic apoptosis. Finally, we showed that the impaired development of mTECs could be partially reversed by decreasing mTORC1 activity via haploinsufficiency of Raptor. Thus, Tsc1 is essential for the homeostasis of mTECs by inhibiting lysosomal-mediated apoptosis through mTORC1-dependent pathways. Key Points TEC-intrinsic ablation of Tsc1 predominantly impairs mTEC with lesser impact on cTEC. Tsc1 is essential for TEC survival by inhibiting lysosomal-mediated apoptosis.