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Introduction: Failure to adequate decidualization leads to adverse pregnancy outcomes including pregnancy loss. Although there are plenty of reports underscoring immune dysfunction as the main cause of abortion in CBA/J females mated with DBA/2 males (CBA/J × DBA/2), little is known about the potential role of impaired endometrial decidualization., Methods: Endometrial stromal cells (ESCs) from CBA/J mice were in-vitro decidualized, and the proteome profile of the secretome was investigated by membrane-based array. CBA/J mice were perfused In-utero with either decidualized ESCs (C×D/D), undecidualized ESCs (C×D/ND), or PBS (C×D/P) 12 days before mating with DBA/2 males. Control mice were not manipulated and were mated with male DBA/2 (C×D) or Balb/c (C×B) mice. On day 13.5 of pregnancy, reproductive parameters were measured. In-vivo tracking of EdU-labeled ESCs was performed using fluorescence microscopy. The frequency of regulatory T cells (Tregs) in paraaortic/renal and inguinal lymph nodes was measured by flow cytometry. The proliferation of pregnant CBA/J splenocytes in response to stimulation with DBA/2 splenocytes was assessed by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) flow cytometry., Results: In C×D/D mice, the resorption rate was reduced to match that seen in the C×B group. Intrauterine perfused ESCs appeared in uterine stroma after 2 days, which remained there for at least 12 days. There was no difference in the number of implantation sites and embryo weight across all groups. The frequency of Tregs in the inguinal lymph nodes was similar across all groups, but it increased in the paraaortic/renal lymph nodes of C×D/D mice to the level found in C×B mice. No significant changes were observed in the proliferation of splenocytes from pregnant C×D/D compared to those of the C×D group in response to stimulation with DBA/2 splenocytes. Decidualization of ESCs was associated with a profound alteration in ESC secretome exemplified by alteration in proteins involved in extracellular matrix (ECM) remodeling, response to inflammation, senescence, and immune cell trafficking., Discussion: Our results showed that the deficiency of Tregs is not the primary driver of abortion in the CBA/J × DBA/2 model and provided evidence that impaired endometrial decidualization probably triggers endometrial immune dysfunction and abortion in this model., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declare that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Zarnani, Zarnani, Maslehat-Lay, Zeynali, Vafaei, Shokri, Vanaki, Soltanghoraee, Mirzadegan, Edalatkhah, Naderi, Sarvari, Attari, Jeddi-Tehrani and Zarnani.)

Unlike conventional CD4+ T cells, which are phenotypically and functionally plastic, invariant NKT (iNKT) cells generally exist in a terminally differentiated state. Naïve CD4+ T cells can acquire alternative epigenetic states in response to different cues, but it remains unclear whether peripheral iNKT cells are epigenetically stable or malleable. Repetitive encounters of liver-resident iNKT cells (LiNKTs) with alpha-galactosylceramide (αGalCer)/CD1d-coated nanoparticles (NPs) can trigger their differentiation into a LiNKT cell subset expressing a T regulatory type 1 (TR1)-like (LiNKTR1) transcriptional signature. Here we dissect the epigenetic underpinnings of the LiNKT-LiNKTR1 conversion as compared to those underlying the peptide-major histocompatibility complex (pMHC)-NP-induced T-follicular helper (TFH)-to-TR1 transdifferentiation process. We show that gene upregulation during the LINKT-to-LiNKTR1 cell conversion is associated with demethylation of gene bodies, inter-genic regions, promoters and distal gene regulatory elements, in the absence of major changes in chromatin exposure or deposition of expression-promoting histone marks. In contrast, the naïve CD4+ T cell-to-TFH differentiation process involves extensive remodeling of the chromatin and the acquisition of a broad repertoire of epigenetic modifications that are then largely inherited by TFH cell-derived TR1 cell progeny. These observations indicate that LiNKT cells are epigenetically malleable and particularly susceptible to gene de-methylation., Competing Interests: PSa is founder, scientific officer and stockholder of Parvus Therapeutics. He is inventor on patents on pMHC-based nanomedicines and receives funding from the company. He also has a consulting agreement with Sanofi. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission., (Copyright © 2024 Montaño, Garnica, Yamanouchi, Moro, Solé, Mondal, Serra, Yang and Santamaria.)

Bleomycin (BLM) induces lung injury, leading to inflammation and pulmonary fibrosis. Regulatory T cells (Tregs) maintain self-tolerance and control host immune responses. However, little is known about their involvement in the pathology of pulmonary fibrosis. Here we show that a unique Treg subset expressing trefoil factor family 1 (Tff1) emerges in the BLM-injured lung. These Tff1-expressing Tregs (Tff1-Tregs) were induced by IL-33. Moreover, although Tff1 ablation in Tregs did not change the pathological condition, selective ablation of Tff1-Tregs using an intersectional genetic method promoted pro-inflammatory features of macrophages in the injured lung and exacerbated the fibrosis. Taken together, our study revealed the presence of a unique Treg subset expressing Tff1 in BLM-injured lungs and their critical role in the injured lung to ameliorate fibrosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Okamoto, Kuratani, Okuzaki, Kamiyama, Kobayashi, Sasai and Yamamoto.)

Our goal is to improve the outcomes of cancer immunotherapy by targeting FOXP3+ T-regulatory (Treg) cells with a next generation of antisense oligonucleotides (ASO), termed FOXP3 AUMsilence ASO. We performed in vitro experiments with human healthy donor PBMC and clinical samples from patients with lung cancer, mesothelioma and melanoma, and tested our approach in vivo using ASO FOXP3 in syngeneic murine cancer models and in humanized mice. ASO FOXP3 had no effects on cell viability or cell division, did not affect expression of other FOXP members, but decreased expression of FOXP3 mRNA in PBMC by 54.9% and in cancer samples by 64.7%, with corresponding 41.0% (PBMC) and 60.0% (cancer) decreases of Treg numbers (all p<0.0001). Hence, intratumoral Treg were more sensitive to the effects of ASO FOXP3 than peripheral blood Tregs. Isolated human Treg, incubated with ASO FOXP3 for 3.5 hours, had significantly impaired suppressive function (66.4%) versus Scramble control. In murine studies, we observed a significant inhibition of tumor growth, while 13.6% (MC38) to 22% (TC1) of tumors were completely resorbed, in conjunction with ~50% decrease of Foxp3 mRNA by qPCR and decreased numbers of intratumoral Tregs. In addition, there were no changes in FOXP3 mRNA expression or in the numbers of Tregs in draining lymph nodes and in spleens of tumor bearing mice, confirming that intratumoral Treg had enhanced sensitivity to ASO FOXP3 in vivo compared to other Treg populations. ASO FOXP3 Treg targeting in vivo and in vitro was accompanied by significant downregulation of multiple exhaustion markers, and by increased expression of perforin and granzyme-B by intratumoral T cells. To conclude, we report that targeting the key Treg transcription factor FOXP3, with ASO FOXP3, has a powerful anti-tumoral effect and enhances T cell response in vitro and in vivo ., Competing Interests: TA is a Senior Scientist, ZB is a Scientist and VA is a Co-founder and Chief Executive Officer of AUM Biotech, LLC, the company that developed, produced and provided Professor Hancock’s laboratory with ASO FOXP3. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Akimova, Wang, Bartosh, Christensen, Eruslanov, Singhal, Aishwarya and Hancock.)

Conventional CD4 pos regulatory T (Treg) cells characterized by expression of the key transcription factor forkhead box P3 (FoxP3) are crucial to control immune responses, thereby maintaining homeostasis and self-tolerance. Within the substantial population of non-conventional T cell receptor (TCR)αβ pos CD4 neg CD8α neg double-negative (dn) T cells of dogs, a novel FoxP3 pos Treg-like subset was described that, similar to conventional CD4 pos Treg cells, is characterized by high expression of CD25. Noteworthy, human and murine TCRαβ pos regulatory dn T cells lack FoxP3. Immunosuppressive capacity of canine dn T cells was hypothesized based on expression of inhibitory molecules (interleukin (IL)-10, cytotoxic T-lymphocyte associated protein 4, CTLA4) . Here, to verify their regulatory function, the dnCD25 pos (enriched for FoxP3 pos Treg-like cells) and the dnCD25 neg fraction, were isolated by fluorescence-activated cell sorting from peripheral blood mononuclear cells (PBMC) of Beagle dogs and analyzed in an in vitro suppression assay in comparison to conventional CD4 pos CD25 pos Treg cells (positive control) and CD4 pos CD25 neg T cells (negative control). Canine dnCD25 pos T cells suppressed the Concanavalin A-driven proliferation of responder PBMC to a similar extent as conventional CD4 pos CD25 pos Treg cells. Albeit to a lesser extent than FoxP3-enriched dn and CD4 pos CD25 pos populations, even dnCD25 neg T cells reduced the proliferation of responder cells. This is remarkable, as dnCD25 neg T cells have a FoxP3 neg phenotype comparable to non-suppressive CD4 pos CD25 neg T cells. Both, CD25 pos and CD25 neg dn T cells, can mediate suppression independent of cell-cell contact and do not require additional signals from CD4 pos CD25 neg T cells to secrete inhibitory factors in contrast to CD4 pos CD25 pos T cells. Neutralization of IL-10 completely abrogated the suppression by dnCD25 pos and CD4 pos CD25 pos Treg cells in a Transwell™ system, while it only partially reduced suppression by dnCD25 neg T cells. Taken together, unique canine non-conventional dnCD25 pos FoxP3 pos Treg-like cells are potent suppressor cells in vitro . Moreover, inhibition of proliferation of responder T cells by the dnCD25 neg fraction indicates suppressive function of a subset of dn T cells even in the absence of FoxP3. The identification of unique immunoregulatory non-conventional dn T cell subpopulations of the dog in vitro is of high relevance, given the immunotherapeutic potential of manipulating regulatory T cell responses in vivo., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Karwig, Moore, Alber and Eschke.)

Histone deacetylases 1 and 2 play a major role in the transcriptional regulation of T-regulatory (Treg) cells via interactions with a myriad of coregulatory factors. Sin3a has been well established as a Hdac1/2 cofactor, while its role within Tregs has not been established. In this study, the effects of conditional deletion of Sin3a within Foxp3+ Tregs were evaluated. Developmental deletion of Sin3a from Foxp3+ Tregs resulted in the rapid onset of fatal autoimmunity. Treg numbers were greatly reduced, while residual Tregs had impaired suppressive function. Mice also showed effector T-cell activation, autoantibody production, and widespread tissue injury. Mechanistically, Sin3a deletion resulted in decreased transcription of Foxp3 with a complete lack of CNS2 CpG demethylation. In addition, Foxp3 protein stability was impaired with an increased ex-Treg population. Thus, Sin3a plays a critical role in the maintenance of Treg identity and function and is essential for the expression and stability of Foxp3., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Christensen, Akimova, Wang, Han, Samanta, Di Giorgio and Hancock.)

Introduction: Ubiquitin-specific proteases (USPs), a large subset of more than 50 deubiquitinase proteins, have recently emerged as promising targets in cancer. However, their role in immune cell regulation, particularly in T cell activation, differentiation, and effector functions, remains largely unexplored., Methods: We utilized a USP28 knockout mouse line to study the effect of USP28 on T cell activation and function, and its role in intestinal inflammation using the dextran sulfate sodium (DSS)-induced colitis model and a series of in vitro assays., Results: Our results show that USP28 exerts protective effects in acute intestinal inflammation. Mechanistically, USP28 knockout mice (USP28-/-) exhibited an increase in total T cells mainly due to an increased CD8+ T cell content. Additionally, USP28 deficiency resulted in early defects in T cell activation and functional changes. Specifically, we observed a reduced expression of IL17 and an increase in inducible regulatory T (iTreg) suppressive functions. Importantly, activated T cells lacking USP28 showed increased STAT5 phosphorylation. Consistent with these findings, these mice exhibited increased susceptibility to acute DSS-induced intestinal inflammation, accompanied by elevated IL22 cytokine levels., Conclusions: Our findings demonstrate that USP28 is essential for T cell functionality and protects mice from acute DSS-induced colitis by regulating STAT5 signaling and IL22 production. As a T cell regulator, USP28 plays a crucial role in immune responses and intestinal health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Le Menn, Pikkarainen, Mennerich, Miroszewska, Kietzmann and Chen.)

Introduction: CD39 plays an important role in the immunoregulation and inhibition of effector cells. It is expressed on immune cells, including Tregs, and on extracellular vesicles (EVs) budding from the plasma membrane. Platelet transfusion may induce alloimmunization against HLA-I antigens, leading to refractoriness to platelet transfusion with severe consequences for patients. Tregs may play a key role in determining whether alloimmunization occurs in patients with hematologic disorders. We hypothesized that CD39 + EVs might play an immunoregulatory role, particularly in the context of platelet transfusions in patients with hematologic disorders. Such alloimmunization leads to the production of alloantibodies and is sensitive to the regulatory action of CD39., Methods: We characterized CD39 + EVs in platelet concentrates by flow cytometry. The absolute numbers and cellular origins of CD39 + EVs were evaluated. We also performed functional tests to evaluate interactions with immune cells and their functions., Results: We found that CD39 + EVs from platelet concentrates had an inhibitory phenotype that could be transferred to the immune cells with which they interacted: CD4 + and CD8 + T lymphocytes (TLs), dendritic cells, monocytes, and B lymphocytes (BLs). Moreover, the concentration of CD39 + EVs in platelet concentrates varied and was very high in 10% of concentrates. The number of these EVs present was determinant for EV-cell interactions. Finally, functional interactions were observed with BLs, CD4 + TLs and CD39 + EVs for immunoglobulin production and lymphoproliferation, with potential implications for the immunological management of patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Delorme, Laguide, Tamagne, Pinheiro, Cagnet, Neyrinck-Leglantier, Khelfa, Cleophax, Pirenne and Vingert.)

Background: Interleukin-17 (IL-17) family cytokines promote protective inflammation for pathogen resistance, but also facilitate autoimmunity and tumor development. A direct signal of IL-17 to regulatory T cells (Tregs) has not been reported and may help explain these dichotomous responses., Methods: We generated a conditional knockout of Il17ra in Tregs by crossing Foxp3-YFP-Cre mice to Il17ra-flox mice ( Il17ra ΔTreg mice). Subsequently, we adoptively transferred bone marrow cells from Il17ra ΔTreg mice to a mouse model of sporadic colorectal cancer ( Cdx2-Cre + / Apc F/+ ), to selectively ablate IL-17 direct signaling on Tregs in colorectal cancer. Single cell RNA sequencing and bulk RNA sequencing were performed on purified Tregs from mouse colorectal tumors, and compared to those of human tumor infiltrating Treg cells., Results: IL-17 Receptor A (IL-17RA) is expressed in Tregs that reside in mouse mesenteric lymph nodes and colon tumors. Ablation of IL-17RA, specifically in Tregs, resulted in increased Th17 cells, and exacerbated tumor development. Mechanistically, tumor-infiltrating Tregs exhibit a unique gene signature that is linked to their activation, maturation, and suppression function, and this signature is in part supported by the direct signaling of IL-17 to Tregs. To study pathways of Treg programming, we found that loss of IL-17RA in tumor Tregs resulted in reduced RNA splicing, and downregulation of several RNA binding proteins that are known to regulate alternative splicing and promote Treg function., Conclusion: IL-17 directly signals to Tregs and promotes their maturation and function. This signaling pathway constitutes a negative feedback loop that controls cancer-promoting inflammation in CRC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Theune, Chen, Theune, Ye, Ménoret, Vella and Wang.)

Foxp3+ regulatory T cells (Foxp3+ Treg) play a role in regulating various types of tumors, but uncertainty still exists regarding the exact mechanism underlying Foxp3+ Treg activation in gastrointestinal malignancies. As of now, research has shown that Foxp3+ Treg expression, altered glucose metabolism, or a hypoxic tumor microenvironment all affect Foxp3+ Treg function in the bodies of tumor patients. Furthermore, it has been demonstrated that post-translational modifications are essential for mature Foxp3 to function properly. Additionally, a considerable number of non-coding RNAs (ncRNAs) have been implicated in the activation of the Foxp3 signaling pathway. These mechanisms regulating Foxp3 may one day serve as potential therapeutic targets for gastrointestinal malignancies. This review primarily focuses on the properties and capabilities of Foxp3 and Foxp3+Treg. It emphasizes the advancement of research on the regulatory mechanisms of Foxp3 in different malignant tumors of the digestive system, providing new insights for the exploration of anticancer treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Ding, Wang, Song, Huo, Chen, Xiang and Liu.)

Introduction: This study examined the impact of 5'-(N- ethylcarboxamido)adenosine (NECA) in the peripheral blood of healthy individuals, those with diabetes mellitus, diabetic retinopathy (DR), and C57BL/6 mice, both in vivo and in vitro ., Methods: Enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM) were used to evaluate the effects of NECA on dendritic cells (DCs) and mouse bone marrow-derived dendritic cells (BMDCs) and the effects of NECA-treated DCs on Treg and Th17 cells. The effect of NECA on the Toll-like receptor (TLR) pathway in DCs was evaluated using polymerase chain reaction (PCR) and western blotting (WB)., Results: FCM and ELISA showed that NECA inhibited the expression of surface markers of DCs and BMDCs, increased anti-inflammatory cytokines and decreased proinflammatory cytokines. PCR and WB showed that NCEA decreased mRNA transcription and protein expression in the TLR-4-MyD88-NF-kβ pathway in DCs and BMDCs. The DR severity in streptozocin (STZ) induced diabetic mice was alleviated. NECA-treated DCs and BMDCs were co-cultivated with CD4+T cells, resulting in modulation of Treg and Th17 differentiation, along with cytokine secretion alterations., Conclusion: NECA could impair DCs' ability to present antigens and mitigate the inflammatory response, thereby alleviating the severity of DR., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Chen, Wang, Xu, Yao, Lei, Zhou and Zheng.)

Macrophages are a rich source of macrophage migration inhibitory factor (MIF). It is well established that macrophages and MIF play a pathogenic role in anti-glomerular basement membrane crescentic glomerulonephritis (anti-GBM CGN). However, whether macrophages mediate anti-GBM CGN via MIF-dependent mechanism remains unexplored, which was investigated in this study by specifically deleting MIF from macrophages in MIF f/f-lysM-cre mice. We found that compared to anti-GBM CGN induced in MIF f/f control mice, conditional ablation of MIF in macrophages significantly suppressed anti-GBM CGN by inhibiting glomerular crescent formation and reducing serum creatinine and proteinuria while improving creatine clearance. Mechanistically, selective MIF depletion in macrophages largely inhibited renal macrophage and T cell recruitment, promoted the polarization of macrophage from M1 towards M2 via the CD74/NF-κB/p38MAPK-dependent mechanism. Unexpectedly, selective depletion of macrophage MIF also significantly promoted Treg while inhibiting Th1 and Th17 immune responses. In summary, MIF produced by macrophages plays a pathogenic role in anti-GBM CGN. Targeting macrophage-derived MIF may represent a novel and promising therapeutic approach for the treatment of immune-mediated kidney diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yang, Li, Huang, Bucala, Xu and Lan.)

The dog is an important companion animal and also serves as model species for human diseases. Given the central role of T cells in immune responses, a basic understanding of canine conventional T cell receptor (TCR)αβ + T cells, comprising CD4 + single-positive (sp) T helper (Th) and CD8α + sp cytotoxic T cell subsets, is available. However, characterization of canine non-conventional TCRαβ + CD4 + CD8α + double-positive (dp) and TCRαβ + CD4 - CD8α - double-negative (dn) T cells is limited. In this study, we performed a comprehensive analysis of canine dp and dn T cells in comparison with their conventional counterparts. TCRαβ + T cells from peripheral blood of healthy dogs were sorted according to their CD4/CD8α phenotype into four populations (i.e. CD4 + sp, CD8α + sp, dp, and dn) and selected surface markers, transcription factors and effector molecules were analyzed ex vivo and after in vitro stimulation by RT-qPCR. Novel characteristics of canine dp T cells were identified, expanding the previously characterized Th1-like phenotype to Th17-like and Th2-like properties. Overall, mRNA expression of various Th cell-associated cytokines (i.e. IFNG, IL17A, IL4, IL13 ) in dp T cells upon stimulation highlights their versatile immunological potential. Furthermore, we demonstrated that the CD4 - CD8α - dn phenotype is stable during in vitro stimulation. Strikingly, dn T cells were found to express highest mRNA levels of type 2 effector cytokines ( IL4, IL5, and IL13 ) upon stimulation. Their strong ability to produce IL-4 was confirmed at the protein level. Upon stimulation, the percentage of IL-4-producing cells was even higher in the non-conventional dn than in the conventional CD4 + sp population. Constitutive transcription of IL1RL1 (encoding IL-33Rα) further supports Th2-like properties within the dn T cell population. These data point to a role of dn T cells in type 2 immunity. In addition, the high potential of dn T cells to transcribe the gene encoding the co-inhibitory receptor CTLA-4 and to produce the inhibitory cytokine IL-10 indicates putative immunosuppressive capacity of this population. In summary, this study reveals important novel aspects of canine non-conventional T cells providing the basis for further studies on their effector and/or regulatory functions to elucidate their role in health and disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Protschka, Di Placido, Moore, Büttner, Alber and Eschke.)

Allergic asthma is a widely prevalent inflammatory condition affecting people across the globe. T cells and their secretory cytokines are central to the pathogenesis of allergic asthma. Here, we have evaluated the anti-inflammatory impact of dimethyl fumarate (DMF) in allergic asthma with more focus on determining its effect on T cell responses in allergic asthma. By utilizing the ovalbumin (OVA)-induced allergic asthma model, we observed that DMF administration reduced the allergic asthma symptoms and IgE levels in the OVA-induced mice model. Histopathological analysis showed that DMF treatment in an OVA-induced animal model eased the inflammation in the nasal and bronchial tissues, with a particular decrease in the infiltration of immune cells. Additionally, RT-qPCR analysis exhibited that treatment of DMF in an OVA-induced model reduced the expression of inflammatory cytokine (IL4, IL13, and IL17) while augmenting anti-inflammatory IL10 and Foxp3 (forkhead box protein 3). Mechanistically, we found that DMF increased the expression of Foxp3 by exacerbating the expression of nuclear factor E2-related factor 2 (Nrf2), and the in-vitro activation of Foxp3+ Tregs leads to an escalated expression of Nrf2. Notably, CD4-specific Nrf2 deletion intensified the allergic asthma symptoms and reduced the in-vitro iTreg differentiation. Meanwhile, DMF failed to exert protective effects on OVA-induced allergic asthma in CD4-specific Nrf2 knock-out mice. Overall, our study illustrates that DMF enhances Nrf2 signaling in T cells to assist the differentiation of Tregs, which could improve the anti-inflammatory immune response in allergic asthma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Cen, Li, Li, Zhang, Riaz, Zhao, Wei and Pan.)

Introduction: CARD11 is a lymphoid lineage-specific scaffold protein regulating the NF-κB activation downstream of the antigen receptor signal pathway. Defective CARD11 function results in abnormal development and differentiation of lymphocytes, especially thymic regulatory T cells (Treg)., Method: In this study, we used patients' samples together with transgenic mouse models carrying pathogenic CARD11 mutations from patients to explore their effects on Treg development. Immunoblotting and a GFP receptor assay were used to evaluate the activation effect of CARD11 mutants on NF-κB signaling. Then the suppressive function of Tregs carrying distinct CARD11 mutations was measured by in vitro suppression assay. Finally, we applied the retroviral transduced bone marrow chimeras to rescue the Treg development in an NF-κB independent manner., Results and Discuss: We found CARD11 mutations causing hyper-activated NF-κB signals also gave rise to compromised Treg development in the thymus, similar to the phenotype in Card11 deficient mice. This observation challenges the previous view that CARD11 regulates Treg lineage dependent on the NF-kB activation. Mechanistic investigations reveal that the noncanonical function CARD11, which negatively regulates the AKT/ FOXO1 signal pathway, is responsible for regulating Treg generation. Moreover, primary immunodeficiency patients carrying CARD11 mutation, which autonomously activates NF-κB, also represented the reduced Treg population in their peripheral blood. Our results propose a new regulatory function of CARD11 and illuminate an NF-κB independent pathway for thymic Treg lineage commitment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hu, Han, Xu, Li, Zhao, Lu, Sun and Wang.)

Regulatory T cells (Tregs) residing in visceral adipose tissue (VAT) play a pivotal role in regulating tissue inflammation and metabolic dysfunction associated with obesity. However, the specific phenotypic and functional characteristics of Tregs in obese VAT, as well as the regulatory mechanisms shaping them, remain elusive. This study demonstrates that obesity selectively reduces Tregs in VAT, characterized by restrained proliferation, heightened PD-1 expression, and diminished ST2 expression. Additionally, obese VAT displays distinctive maturation of dendritic cells (DCs), marked by elevated expressions of MHC-II, CD86, and PD-L1, which are inversely correlated with VAT Tregs. In an in vitro co-culture experiment, only obese VAT DCs, not macrophages or DCs from subcutaneous adipose tissue (SAT) and spleen, result in decreased Treg differentiation and proliferation. Furthermore, Tregs differentiated by obese VAT DCs exhibit distinct characteristics resembling those of Tregs in obese VAT, such as reduced ST2 and IL-10 expression. Mechanistically, obesity lowers IL-33 production in VAT DCs, contributing to the diminished Treg differentiation. These findings collectively underscore the critical role of VAT DCs in modulating Treg generation and shaping Treg phenotype and function during obesity, potentially contributing to the regulation of VAT Treg populations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Soedono, Sharlene, Vo, Averia, Rosalie, Lee and Cho.)

Neuropilin-1 (Nrp1), a transmembrane protein expressed on CD4 + T cells, is mostly studied in the context of regulatory T cell (Treg) function. More recently, there is increasing evidence that Nrp1 is also highly expressed on activated effector T cells and that increases in these Nrp1-expressing CD4 + T cells correspond with immunopathology across several T cell-dependent disease models. Thus, Nrp1 may be implicated in the identification and function of immunopathologic T cells. Nrp1 downregulation in CD4 + T cells is one of the strongest transcriptional changes in response to immunoregulatory compounds that act though the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. To better understand the link between AhR and Nrp1 expression on CD4 + T cells, Nrp1 expression was assessed in vivo and in vitro following AhR ligand treatment. In the current study, we identified that the percentage of Nrp1 expressing CD4 + T cells increases over the course of activation and proliferation in vivo . The actively dividing Nrp1 + Foxp3 - cells express the classic effector phenotype of CD44 hi CD45RB lo , and the increase in Nrp1 + Foxp3 - cells is prevented by AhR activation. In contrast, Nrp1 expression is not modulated by AhR activation in non-proliferating CD4 + T cells. The downregulation of Nrp1 on CD4 + T cells was recapitulated in vitro in cells isolated from C57BL/6 and NOD (non-obese diabetic) mice. CD4 + Foxp3 - cells expressing CD25, stimulated with IL-2, or differentiated into Th1 cells, were particularly sensitive to AhR-mediated inhibition of Nrp1 upregulation. IL-2 was necessary for AhR-dependent downregulation of Nrp1 expression both in vitro and in vivo . Collectively, the data demonstrate that Nrp1 is a CD4 + T cell activation marker and that regulation of Nrp1 could be a previously undescribed mechanism by which AhR ligands modulate effector CD4 + T cell responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sandoval, Malany, Thongphanh, Martinez, Goodson, Souza, Lin, Sweeney, Pennington, Lein, Kerkvliet and Ehrlich.)

T Regulatory type-1 (TR1) cells represent an immunosuppressive T cell subset, discovered over 25 years ago, that produces high levels of interleukin-10 (IL-10) but, unlike its FoxP3+ T regulatory (Treg) cell counterpart, does not express FoxP3 or CD25. Experimental evidence generated over the last few years has exposed a promising role for TR1 cells as targets of therapeutic intervention in immune-mediated diseases. The discovery of cell surface markers capable of distinguishing these cells from related T cell types and the application of next generation sequencing techniques to defining their transcriptional make-up have enabled a more accurate description of this T cell population. However, the developmental biology of TR1 cells has long remained elusive, in particular the identity of the cell type(s) giving rise to bona fide TR1 cells in vivo . Here, we review the fundamental phenotypic, transcriptional and functional properties of this T cell subset, and summarize recent lines of evidence shedding light into its ontogeny., Competing Interests: PS is founder, scientific officer and stock holder of Parvus Therapeutics. He is inventor on patents on pMHC-based nanomedicines and receives funding from the company. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision, (Copyright © 2023 Angelats and Santamaria.)

Introduction: Placental trophoblasts contribute to regulatory T (Treg) function via the programmed cell death-1 (PD-1)/PD-1 ligand 1 (PD-L1) pathway during normal pregnancy. Decreased expression of PD-L1 in trophoblasts was closely associated with Treg deficiency in the development of pregnancy failure. Thus, targeting PD-L1 might be a novel therapy to prevent pregnancy loss. However, the mechanisms for modulating the expression of PD-L1 in trophoblasts are an enigma., Methods: The proportion of decidual Treg cells, and the profile of decidual macrophages (DMs) sampled from women with normal pregnancy (NP) and recurrent miscarriage (RM) were evaluated by flow cytometry. The expression of Yin and Yang 1 protein (YY1) and PD-L1 in human villous were measured by Immunohistochemistry (IHC), qRT-PCR and western blot. The determination of soluble PD-L1 (sPD-L1) in serum from NP and RM, and trophoblast conditioned media (TCM) was performed by the PD-L1 SimpleStep ELISA kit. Knockdown of YY1 was processed in the human trophoblast derived cell lines, HTR-8 and Bewo, with siYY1 transfection. Peripheral naïve CD4 + T cells were isolated from women with NP for the in vitro culture. The percentages of Treg cells differentiated from peripheral naïve CD4 + T cells were measured by flow cytometry. The interaction between YY1 and CD274 was proved by CHIP. The expression of inducible nitric oxide synthase (iNOS) in decidua was evaluated by IHC. The level of NO in serum from women with NP and RM was determined by the Griess reagent system. The effects of NO on YY1 were determined by the in vitro culture of HTR-8 cells with the NO donor, SNAP. The in vivo model comprising twelve pregnant mice and underwent different treatment. The percentages of Treg cells in murine uterus were measured by flow cytometry. Similarly, Western blot and IHC were performed to determine the expression of YY1 and PD-L1 in murine placenta., Results: Decreased expression of YY1 and PD-L1 in trophoblasts and lower proportion of decidual Treg cells were observed in patients with RM. Knockdown of YY1 contributes to a lower expression of YY1 and PD-L1. Soluble PD-L1 in the supernatant from HTR-8 cells was also decreased with siYY1 administration. Lower Treg differentiation was observed in the presence of supernatant from HTR-8 cells treated with siYY1. CHIP analysis revealed that endogenous YY1 directly occupied the promoter region of the CD274 (PD-L1) gene. Accompanied with increased M1 DMs, higher NO was observed in serum sampled from patients with RM. In the presence of Reduced expression of YY1 and PD-L1 was observed in HTR-8 cells with the treatment of SNAP. Furthermore, less Treg differentiation was observed with SNAP treated TCM. Moreover, our in vivo data found that YY1 deficiency was associated with decreased PD-L1, which further resulting in less Treg differentiation and Treg deficiency at the maternal-fetal interface and increased embryo loss., Discussion: Our work found the modulatory capacity of YY1 on PD-L1 in trophoblasts during early pregnancy. Furthermore, reduced YY1 was supposed resulting from higher levels of NO produced from the M1 DMs in RM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Wang, Qiu, Jiang, Wu, Mei and Sun.)

Introduction: T cell-dependent inflammatory response with the upregulation of helper 17 T cells (Th17) and the downregulation of regulatory T cells (Treg) accompanied by the increased production of tumor necrosis alpha (TNFa) is characteristic of inflammatory bowel diseases (IBD). Modulation of T cell response may alleviate the inflammation thus reduce intestinal damage. Poly(ADP-ribose) polymerase-2 (PARP2) plays role in the development, differentiation and reactivity of T cell subpopulations. Our aim was to investigate the potential beneficial effect of T cell-specific PARP2 downregulation in the lipopolysaccharide (LPS) induced inflammatory response of the cecum and the colon., Methods: Low-dose LPS was injected intraperitoneally to induce local inflammatory response, characterized by increased TNFa production, in control (CD4Cre; PARP2+/+) and T cell-specific conditional PARP2 knockout (CD4Cre; PARP2f/f) mice. TNFa, IL-1b, IL-17 levels were measured by ELISA, oxidative-nitrative stress was estimated by immunohistochemistry, while PARP1 activity, p38 MAPK and ERK phosphorylation, and NF-kB expression in large intestine tissue samples were examined by Western-blot. Systemic & local T cell subpopulation; Th17 and Treg alterations were also investigated using flowcytometry and immunohistochemistry., Results: In control animals, LPS induced intestinal inflammation with increased TNFa production, while no significant elevation of TNFa production was observed in T cell-specific PARP2 knockout animals. The absence of LPS-induced elevation in TNFa levels was accompanied by the absence of IL-1b elevation and the suppression of IL-17 production, showing markedly reduced inflammatory response. The increase in oxidative-nitrative stress and PARP1-activation was also absent in these tissues together with altered ERK and NF-kB activation. An increase in the number of the anti-inflammatory Treg cells in the intestinal mucosa was observed in these animals, together with the reduction of Treg count in the peripheral circulation., Discussion: Our results confirmed that T cell-specific PARP2 downregulation ameliorated LPS-induced colitis. The dampened TNFa production, decreased IL-17 production and the increased intestinal regulatory T cell number after LPS treatment may be also beneficial during inflammatory processes seen in IBD. By reducing oxidative-nitrative stress and PARP1 activation, T cell-specific PARP2 downregulation may also alleviate intestinal tissue damage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bencsics, Bányai, Ke, Csépányi-Kömi, Sasvári, Dantzer, Hanini, Benkő and Horváth.)

Foxp3 is regarded as the major transcription factor for T regulatory (T reg ) cells and expression of Foxp3 is used to identify and quantitate Treg cells in mouse models. However, several studies have demonstrated that human CD4 + T conventional (T conv ) cells activated in vitro by T cell receptor (TCR) stimulation can express Foxp3. This observation has raised doubt as to the suitability of Foxp3 as a T reg marker in man. Helios, a member of the Ikaros gene family, has been shown to be expressed by 80-90% of human Foxp3 + T reg cells and can potentially serve as a marker of human T reg . Here, we confirm that Foxp3 expression is readily upregulated by T conv upon TCR stimulation in vitro , while Helios expression is not altered. More importantly, we show that Foxp3 expression is not elevated by stimulation of hT conv in a humanized mouse model of graft versus host disease (GVHD) and in patients with a wide variety of acute and chronic inflammatory diseases including sickle cell disease, acute and chronic GVHD, systemic lupus erythematosus, as well as critical COVID-19. In all patients studied, an excellent correlation was observed between the percentage of CD4 + T cells expressing Foxp3 and the percentage expressing Helios. Taken together, these studies demonstrate that Foxp3 is not induced upon T conv cell activation in vivo and that Foxp3 expression alone can be used to quantitate T reg cells in humans. Nevertheless, the combined use of Foxp3 and Helios expression provides a more reliable approach for the characterization of T reg in humans., Competing Interests: MB is employed by the company Janssen Pharmaceuticals. AG is employed by the company Miltenyi Biotech. CO is employed by Axle Informatics. Studies unrelated to the present manuscript in the Shevach Laboratory (LM, MJ, ES, and MB) are supported by CRADAs from Janssen Pharmaceuticals and Boehringer Ingelheim Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Morina, Jones, Oguz, Kaplan, Gangaplara, Fitzhugh, Kanakry, Shevach and Buszko.)

Background: Allergic airway disease (AAD) is a chronic disease characterized by airway inflammation, bronchoconstriction, and hyperresponsiveness. Although exogenous interleukin-10 (IL-10) alleviates allergic inflammation, it has a short half-life in vivo . Cell membrane-coated nanomaterials have been shown to protect therapeutic payloads and increase therapeutic efficacy., Objective: This study was aimed at investigating the efficacy of a novel macrophage-based nanoparticle drug for the treatment of house dust mite (HDM)-induced allergic airway diseases., Methods: IL-10-poly (lactic-co-glycolic acid (PLGA) nanoparticles were encapsulated in alveolar macrophage cell membranes. An allergic airway disease mouse model was established by repeated inhalation of HDM extracts. The mice were treated with free IL-10, IL-10-PLGA nanoparticles (IL10-NP), or IL-10-alveolar macrophage cell membrane-coated nanoparticles (IL10-AMNP). The therapeutic effects were evaluated by measuring airway hyperresponsiveness, lung inflammation, cytokine levels, and regulatory T cells (Treg)- T-helper 17 (Th17) cell balance., Results: Compared to free IL-10, IL10-AMNP significantly reduced airway hyperresponsiveness and T-helper 2 (Th2)/Th17 cytokines and inhibited neutrophilia and eosinophilia recruitment into the airways of HDM-induced mouse models. Additionally, the balance between Tregs and Th17 cells was significantly improved in groups treated with IL10-AMNP., Conclusion: This study demonstrated that PLGA nanoparticle cores coated with alveolar macrophage cell membranes can effectively deliver therapeutic cytokines to the lungs and improve the homeostatic balance between Tregs and Th17 cells. These findings suggest that macrophage-based nanoparticle drugs represent a promising approach for treating allergic airway diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li and Yin.)

Introduction: Most T lymphocytes, including regulatory T cells, express the CD27 costimulatory receptor in steady state conditions. There is evidence that CD27 engagement on conventional T lymphocytes favors the development of Th1 and cytotoxic responses in mice and humans, but the impact on the regulatory lineage is unknown., Methods: In this report, we examined the effect of constitutive CD27 engagement on both regulatory and conventional CD4 + T cells in vivo , in the absence of intentional antigenic stimulation., Results: Our data show that both T cell subsets polarize into type 1 Tconvs or Tregs, characterized by cell activation, cytokine production, response to IFN-γ and CXCR3-dependent migration to inflammatory sites. Transfer experiments suggest that CD27 engagement triggers Treg activation in a cell autonomous fashion., Conclusion: We conclude that CD27 may regulate the development of Th1 immunity in peripheral tissues as well as the subsequent switch of the effector response into long-term memory., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bowakim-Anta, Acolty, Azouz, Yagita, Leo, Goriely, Oldenhove and Moser.)

At homeostasis, a substantial proportion of Foxp3 + T regulatory cells (T regs ) have an activated phenotype associated with enhanced TCR signals and these effector T reg cells (eT regs ) co-express elevated levels of PD-1 and CTLA-4. Short term in vivo blockade of the PD-1 or CTLA-4 pathways results in increased eT reg populations, while combination blockade of both pathways had an additive effect. Mechanistically, combination blockade resulted in a reduction of suppressive phospho-SHP2 Y580 in eT reg cells which was associated with increased proliferation, enhanced production of IL-10, and reduced dendritic cell and macrophage expression of CD80 and MHC-II. Thus, at homeostasis, PD-1 and CTLA-4 function additively to regulate eT reg function and the ability to target these pathways in T reg cells may be useful to modulate inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pereira, Lanzar, Clark, Hart, Douglas, Shallberg, O’Dea, Christian and Hunter.)

The development of new strategies based on the use of Tr1 cells has taken relevance to induce long-term tolerance, especially in the context of allogeneic stem cell transplantation. Although Tr1 cells are currently identified by the co-expression of CD49b and LAG-3 and high production of interleukin 10 (IL-10), recent studies have shown the need for a more exhaustive characterization, including co-inhibitory and chemokines receptors expression, to ensure bona fide Tr1 cells to be used as cell therapy in solid organ transplantation. Moreover, the proinflammatory environment induced by the allograft could affect the suppressive function of Treg cells, therefore stability of Tr1 cells needs to be further investigated. Here, we establish a new protocol that allows long-term in vitro expansion of highly purified expanded allospecific Tr1 ( Exp-allo Tr1). Our expanded Tr1 cell population becomes highly enriched in IL-10 producers (> 90%) and maintains high expression of CD49b and LAG-3, as well as the co-inhibitory receptors PD-1, CTLA-4, TIM-3, TIGIT and CD39. Most importantly, high dimensional analysis of Exp-allo Tr1 demonstrated a specific expression profile that distinguishes them from activated conventional T cells (T conv), showing overexpression of IL-10, CD39, CTLA-4 and LAG-3. On the other hand, Exp-allo Tr1 expressed a chemokine receptor profile relevant for allograft homing and tolerance induction including CCR2, CCR4, CCR5 and CXCR3, but lower levels of CCR7. Interestingly, Exp-allo Tr1 efficiently suppressed allospecific but not third-party T cell responses even after being expanded in the presence of proinflammatory cytokines for two extra weeks, supporting their functional stability. In summary, we demonstrate for the first time that highly purified allospecific Tr1 ( Allo Tr1) cells can be efficiently expanded maintaining a stable phenotype and suppressive function with homing potential to the allograft, so they may be considered as promising therapeutic tools for solid organ transplantation., Competing Interests: CA-S was employed by Accellix. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2023 Arteaga-Cruz, Cortés-Hernández, Alvarez-Salazar, Rosas-Cortina, Aguilera-Sandoval, Morales-Buenrostro, Alberú-Gómez and Soldevila.)

Mast cells (MCs) are innate immune cells with a versatile set of functionalities, enabling them to orchestrate immune responses in various ways. Aside from their known role in allergy, they also partake in both allograft tolerance and rejection through interaction with regulatory T cells, effector T cells, B cells and degranulation of cytokines and other mediators. MC mediators have both pro- and anti-inflammatory actions, but overall lean towards pro-fibrotic pathways. Paradoxically, they are also seen as having potential protective effects in tissue remodeling post-injury. This manuscript elaborates on current knowledge of the functional diversity of mast cells in kidney transplants, combining theory and practice into a MC model stipulating both protective and harmful capabilities in the kidney transplant setting., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 van der Elst, Varol, Hermans, Baan, Duong-van Huyen, Hesselink, Kramann, Rabant, Reinders, von der Thüsen, van den Bosch and Clahsen-van Groningen.)

The organ-specific microbiome plays a crucial role in tissue homeostasis, among other things by inducing regulatory T cells (Treg). This applies also to the skin and in this setting short chain fatty acids (SCFA) are relevant. It was demonstrated that topical application of SCFA controls the inflammatory response in the psoriasis-like imiquimod (IMQ)-induced murine skin inflammation model. Since SCFA signal via HCA2, a G-protein coupled receptor, and HCA2 expression is reduced in human lesional psoriatic skin, we studied the effect of HCA2 in this model. HCA2 knock-out (HCA2-KO) mice reacted to IMQ with stronger inflammation, presumably due to an impaired function of Treg. Surprisingly, injection of Treg from HCA2-KO mice even enhanced the IMQ reaction, suggesting that in the absence of HCA2 Treg switch from a suppressive into a proinflammatory type. HCA2-KO mice differed in the composition of the skin microbiome from wild type mice. Co-housing reversed the exaggerated response to IMQ and prevented the alteration of Treg, implying that the microbiome dictates the outcome of the inflammatory reaction. The switch of Treg into a proinflammatory type in HCA2-KO mice could be a downstream phenomenon. This opens the opportunity to reduce the inflammatory tendency in psoriasis by altering the skin microbiome., Competing Interests: SR-J has acted as a consultant and speaker for AbbVie, Chugai, Genentech Roche, Pfizer and Sanofi. He also declares that he is an inventor on patents owned by CONARIS Research Institute, which develops the sgp130Fc protein Olamkicept together with the companies Ferring and I-Mab. He has stock ownership in CONARIS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schwarz, Philippsen, Piticchio, Hartmann, Häsler, Rose-John and Schwarz.)

Recent studies have revealed that tumor-associated macrophages are the most abundant stromal cells in the tumor microenvironment and play an important role in tumor initiation and progression. Furthermore, the proportion of macrophages in the tumor microenvironment is associated with the prognosis of patients with cancer. Tumor-associated macrophages can polarize into anti-tumorigenic phenotype (M1) and pro-tumorigenic phenotype (M2) by the stimulation of T-helper 1 and T-helper 2 cells respectively, and then exert opposite effects on tumor progression. Besides, there also is wide communication between tumor-associated macrophages and other immune compositions, such as cytotoxic T cells, regulatory T cells, cancer-associated fibroblasts, neutrophils and so on. Furthermore, the crosstalk between tumor-associated macrophages and other immune cells greatly influences tumor development and treatment outcomes. Notably, many functional molecules and signaling pathways have been found to participate in the interactions between tumor-associated macrophages and other immune cells and can be targeted to regulate tumor progression. Therefore, regulating these interactions and CAR-M therapy are considered to be novel immunotherapeutic pathways for the treatment of malignant tumors. In this review, we summarized the interactions between tumor-associated macrophages and other immune compositions in the tumor microenvironment and the underlying molecular mechanisms and analyzed the possibility to block or eradicate cancer by regulating tumor-associated macrophage-related tumor immune microenvironment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Jiang, Wei, Wang and Li.)

Background and Aim: Alkaline sphingomyelinase (NPP7) is expressed by intestinal epithelial cells and is crucial for the digestion of dietary sphingomyelin. NPP7 also inactivates proinflammatory mediators including platelet-activating factor and lysophosphatidylcholine. The aim of this study was to examine a potential role for NPP7 in the homeostasis of the intestinal immune system., Methods: We quantified the numbers of B-lymphocytes, plasma cells, T-lymphocytes including regulatory T-lymphocytes (T regs ), natural killer cells, dendritic cells, macrophages, and neutrophils, in the small and large intestines, the mesenteric lymph nodes and the spleens of heterozygous and homozygous NPP7 knockout (KO) and wildtype (WT) mice. Tissues were examined by immunohistochemistry and stainings quantified using computerized image analysis., Results: The numbers of both small and large intestinal CD3ε + , CD4 + , and CD8α + T-lymphocytes were significantly higher in NPP7 KO compared to WT mice (with a dose-response relationship in the large intestine), whereas T reg numbers were unchanged, and dendritic cell numbers reduced. In contrast, the numbers of CD3ε + and CD4 + T-lymphocytes in mesenteric lymph nodes were significantly reduced in NPP7 KO mice, while no differences were observed in spleens. The numbers of B-lymphocytes, plasma cells, natural killer cells, macrophages, and neutrophils were similar between genotypes., Conclusion: NPP7 contributes to the regulation of dendritic cell and T-lymphocyte numbers in mesenteric lymph nodes and both the small and large intestines, thus playing a role in the homeostasis of gut immunity. Although it is likely that the downstream effects of NPP7 activity involve the sphingomyelin metabolites ceramide and spingosine-1-phosphate, the exact mechanisms behind this regulatory function of NPP7 need to be addressed in future studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Alyamani, Kadivar, Erjefält, Johansson-Lindbom, Duan, Nilsson and Marsal.)

Background: Regulatory T cells (Tregs) have been found to play crucial roles in immune tolerance. However, the status of Tregs in refractory rheumatoid arthritis (RA) is still unclear. Moreover, low-dose interleukin-2 (IL-2) has been reported to selectively promote the expansion of Tregs. This study investigated the status of CD4 + Tregs and low-dose IL-2 therapy in patients with refractory RA., Methods: The absolute number of CD4 + CD25 + FOXP3 + Treg (CD4 Treg), CD4 + IL17 + T (Th17), and other subsets in peripheral blood (PB) from 41 patients with refractory RA and 40 healthy donors was characterized by flow cytometry combined with an internal microsphere counting standard. Twenty-six patients with refractory RA were treated with daily subcutaneous injections of 0.5 million IU of human IL-2 for five consecutive days. Then, its effects on CD4 Treg and Th17 cells in PB were analyzed., Results: A decrease in the absolute number of PB CD4 Tregs rather than the increase in the number of Th17 was found to contribute to an imbalance between Th17 and CD4 Tregs in these patients, suggesting an essential role of CD4 Tregs in sustained high disease activity. Low-dose IL-2 selectively increased the number of CD4 Tregs and rebalanced the ratio of Th17 and CD4 Tregs, leading to increased clinical symptom remission without the observed side effects., Conclusions: An absolute decrease of PB CD4 Tregs in patients with refractory RA was associated with continuing disease activation but not the increase of Th17 cells. Low-dose IL-2, a potential therapeutic candidate, restored decreased CD4 Tregs and promoted the rapid remission of patients with refractory RA without overtreatment and the observed side effects., Clinical Trial Registration: http://www.chictr.org.cn/showproj.aspx?proj=13909, identifier ChiCTR-INR-16009546., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Zhang, Chang, Cheng, Jiang, Su, Zhang, Luo and Li.)

Objective: In order to determine whether the immune balance of T helper 17(Th17)/regulatory T(Treg) is related to the pathogenesis of idiopathic retroperitoneal fibrosis (IRPF), we analyzed the differences in peripheral blood lymphocytes, CD4+T cell subsets and cytokines between patients with IRPF and healthy people to clarify the CD4+T cell subsets, especially Treg cell subsets, and the role of cytokines in the pathogenesis of IRPF., Methods: This study included 22 patients with IRPF, 36 patients with IgG4-related diseases (IgG4-RD) without retroperitoneal fibrosis (RPF), and 28 healthy controls. The absolute numbers and percentage of peripheral blood lymphocyte subsets and CD4+T cell subsets in each group were detected by flow cytometry, and the serum cytokine level was detected by flow cytometric bead array (CBA)., Results: Compared with the healthy group, the absolute value of B cells in peripheral blood of IRPF patients was significantly decreased, and T, natural killer (NK), CD4+ and CD8+ were not significantly abnormal. The absolute numbers of Th2 cells were lower than healthy group(p=0.043). In particular, the absolute numbers of Treg cells were significantly lower than healthy group(p<0.001), while the absolute numbers of Th17 cells increased(p=0.682). Th17/Treg was significantly higher than healthy group (p< 0.001). Cytokine analysis showed that the level of interleukin (IL)-4 in IRPF patients was higher than healthy group(p=0.011), IL-6, IL-10, IL-17, TNF-α and IFN-γ were significantly higher than healthy group (all p<0.001). Receiver operating characteristic (ROC) curves showed that IL-10 and TNF-α could distinguish bilateral ureteral dilatation in IRPF patients, with areas under the ROC curve (AUCs) of 0.813 (95% CI:0.607-1.000, p=0.026) and 0.950 (95% CI:0.856-1.000, p=0.001), respectively. IL-6 could distinguish bilateral ureteral obstruction, with an AUC of 0.861 (95% CI: 0.682-1.000, p=0.015)., Conclusions: Our study showed that IRPF patients had reduced Treg cells and indeed had Th17/Treg imbalance, which may be related to the pathogenesis of the disease. The levels of IL-6, IL-10 and TNF-α appear to be associated with the progression of IRPF., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Yan, Wang, Xie, Jiang, Zhao, Gao, Li and Wang.)

Objectives: Cuproptosis is a recently discovered form of programmed cell death; however, its role in ulcerative colitis (UC) remains a void., Methods: Three gene expression profiles were acquired from the GEO database. Subsequently, the single sample gene set enrichment analysis (ssGSEA) was performed to identify the immune infiltration characteristics of UC. Correlation analysis between cuproptosis and immune infiltration was further conducted, and the cuproptosis-related genes were applied to construct a UC diagnostic model. Subsequently, analysis results of microarray data were experimentally validated by DSS-induced colitis in mice. Finally, therapeutic agents for the cuproptosis-related genes were screened owing to the gaping field of therapeutic agents on cuproptosis., Results: Three gene expression profiles with 343 samples (290 UC and 53 healthy samples) were included. Immune infiltration revealed that UC patients had a higher level of DCs, B cells, CD8 + T cells, iDCs, Macrophages, neutrophils, pDCs, T helper cells, Tfh, Th1 cells, Th2 cells, TIL and Treg than normal subjects. Moreover, almost all cuproptosis-related genes were significantly negatively associated with immune infiltration in UC patients. The risk prediction model based on cuproptosis-related genes showed an excellent discrimination for UC. Animal experiments revealed significant alterations in genes essential for cuproptosis between DSS-induced colitis mice and healthy controls, providing experimental validation for the analysis results of microarray data. Further analysis revealed that latamoxef, vitinoin, clomipramine, chlorzoxazone, glibenclamide, pyruvic acid, clindamycin, medrysone, caspan, and flavin adenine dinucleotide might be the target agents for cuproptosis-related genes., Conclusions: In conclusion, cuproptosis was significantly associated with immune infiltration in UC, and the cuproptosis-related genes showed an excellent discrimination for UC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Huang, Zhang, Wang, Zhang and Tang.)

N6-methyladenosine (m6A), the most prevalent form of internal mRNA modification, is extensively involved in Treg cells differentiation and function. However, the involvement of m6A in functional Treg cells for transplantation tolerance remains to be elucidated. By using an experimental transplantation mouse model, we found that m6A levels in Treg cells were altered during the induction of transplant tolerance by performing a dot blotting assay. Subsequently, we used the heterogenic Treg-specific Mettl14 knockout mice (Foxp3-Mettl14 f/+ cKO) to reduce METTL14 expression and performed islets allograft transplantation. Our result revealed that reduced expression of METTL14 prevented Treg cells expansion and promoted the infiltration of CD4 + and CD8 + T cells around the allograft, which led to rapid allograft rejection in Foxp3-Mettl14 f/+ cKO mice. The expression of regulatory cytokines including IL-10 and TGF-β was significantly decreased in Foxp3-Mettl14 f/+ cKO mice, and the suppressive function of Treg cells was also abrogated. In addition, an analysis of RNA-seq data revealed that the SOCS family (SOCS1, SOCS2 and SOCS3) is the subsequent signaling pathway affected by the METTL14 mediated m6A modification in Treg cells to modulate the suppressive function after transplantation. Taken together, our study showed for the first time that the METTL14-mediated m6A modification is essential for the suppressive function of Treg cells in transplantation and may serve as a regulatory element of Treg cell-based therapy in transplant medicine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Yuan, Zhou, Cui, Teng, Huang, Yuan, Zhang, Yang and Zhao.)

The role of T cells in appendage regeneration remains unclear. In this study, we revealed an important role for regulatory T cells (Tregs), a subset of T cells that regulate tolerance and tissue repair, in the epimorphic regeneration of zebrafish caudal fin tissue. Upon amputation, fin tissue-resident Tregs infiltrate into the blastema, a population of progenitor cells that produce new fin tissues. Conditional genetic ablation of Tregs attenuates blastemal cell proliferation during fin regeneration. Blastema-infiltrating Tregs upregulate the expression of igf2a and igf2b , and pharmacological activation of IGF signaling restores blastemal proliferation in Treg-ablated zebrafish. These findings further extend our understandings of Treg function in tissue regeneration and repair., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hui, Sugimoto, Sheng and Kikuchi.)

Background/aims: Hepatocellular carcinoma (HCC), accounting for 75-85% of primary liver cancer cases, is the third leading cause of cancer-related death worldwide. The purpose of this research was to examine the tumor immune microenvironment (TIME) in HCC., Methods: We investigated the HCC TIME by integrated analysis of single-cell and bulk-tissue sequencing data to reveal the landscape of major immune cell types., Results: Regulatory T(Treg) cells were found to be specifically distributed in the TIME of HCC. Several immune checkpoints, including TNFRSF4, TIGIT and CTLA4, were found to be uniquely overexpressed in Treg cells, and the glycolysis/gluconeogenesis pathway was enriched in Treg cells. We also discovered the presence of two NK-cell subsets with different cytotoxic capacities, one in an activated state with antitumor effects and another with an exhausted status. In addition, memory B cells in HCC were found to exist in a unique state, with high proliferation, low differentiation, and low activity, which was induced by overexpression of PRAP1 and activation of the MIF-CD74 axis., Conclusions: We revealed the TIME landscape in HCC, highlighting the heterogeneity of major immune cell types and their potential mechanisms in the formation of an immunosuppressive environment. Hence, blocking the formation of the TIME could be a useful therapeutic strategy for HCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bai, Chen, Cheng, Li, Chi, Zhang, Zhang, Tang, Zhao, Ang and Zhang.)

The immunosuppressive regulatory T (Treg) cells exert emerging effects on adipose tissue homeostasis and systemic metabolism. However, the metabolic regulation and effector mechanisms of Treg cells in coping with obesogenic insults are not fully understood. We have previously established an indispensable role of the O-linked N-Acetylglucosamine (O-GlcNAc) signaling in maintaining Treg cell identity and promoting Treg suppressor function, via STAT5 O-GlcNAcylation and activation. Here, we investigate the O-GlcNAc transferase (OGT)-STAT5 axis in driving the immunomodulatory function of Treg cells for metabolic homeostasis. Treg cell-specific OGT deficiency renders mice more vulnerable to high-fat diet (HFD)-induced adiposity and insulin resistance. Conversely, constitutive STAT5 activation in Treg cells confers protection against adipose tissue expansion and impaired glucose and insulin metabolism upon HFD feeding, in part by suppressing adipose lipid uptake and redistributing systemic iron storage. Treg cell function can be augmented by targeting the OGT-STAT5 axis to combat obesity and related metabolic disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Salgado, Liu, Moazzami, Hogquist, Farrar and Ruan.)

Background: Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) have been increasingly proved as promising immunomodulators against some autoimmune disorders. However, the possible effect and the underlying mechanism of MSC-sEVs in autoimmune dry eye have been rarely studied., Methods: Small extracellular vesicles from human umbilical cord mesenchymal stem cells (hUC-MSC-sEVs) were subconjunctivally injected to rabbit dry eye model, and their preventive or therapeutical effects were assessed by recording the clinical and histological scores. Quantitative real-time PCR (Q-PCR), western blot and flow cytometry were performed to evaluate the immunomodulatory effects of hUC-MSC-sEVs on macrophages and T regulatory cells (Tregs) both in vivo and in vitro , and the in vitro T cell proliferation was detected by Bromodeoxyuridine (BrdU) assay. In addition, high expression of miR-100-5p in hUC-MSC-sEVs was identified by Q-PCR, and the functional role of sEVs-miR-100-5p on macrophages was explored by a series of co-culture experiments using sEVs derived from hUC-MSCs transfected with miR-100-5p inhibitor., Results: We firstly demonstrated that hUC-MSC-sEVs had the preventive and therapeutical effects on rabbit autoimmune dacryoadenitis, an animal model of Sjögren's syndrome (SS) dry eye. Further investigation revealed that hUC-MSC-sEVs administration effectively elicited macrophages into an anti-inflammatory M2 phenotype and elevated the proportion of Tregs both in vivo and in vitro , which contributed to reduced inflammation and improved tissue damage. Importantly, hUC-MSC-sEVs-educated macrophages with M2-like phenotype exhibited strong capacity to inhibit CD4+ T cell proliferation and promote Treg generation in vitro . Mechanistically, miR-100-5p was highly enriched in hUC-MSC-sEVs, and knockdown of miR-100-5p in hUC-MSC-sEVs partially blunted the promotion of hUC-MSC-sEVs on M2 macrophage polarization and even attenuated the effect of hUC-MSC-sEVs-educated macrophages on T cell suppression and Treg expansion., Conclusion: Our data indicated that hUC-MSC-sEVs alleviated autoimmune dacryoadenitis by promoting M2 macrophage polarization and Treg generation possibly through shuttling miR-100-5p. This study sheds new light on the application of MSC-sEVs as a promising therapeutic method for SS dry eye., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Gao, Zhao, Du, Ma, Nian and Wei.)

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a very common urological disorder and has been gradually regarded as an immune-mediated disease. Multiple studies have indicated that the gut microflora plays a pivotal part in immune homeostasis and autoimmune disorder development. However, whether the gut microflora affects the CP/CPPS, and the underlying mechanism behind them remain unclear. Here, we built an experimental autoimmune prostatitis (EAP) mouse model by subcutaneous immunity and identified that its Th17/Treg frequency was imbalanced. Using fecal 16s rRNA sequencing and untargeted/targeted metabolomics, we discovered that the diversity and relative abundance of gut microflora and their metabolites were obviously different between the control and the EAP group. Propionic acid, a kind of short-chain fatty acid (SCFA), was decreased in EAP mice compared to that in controls, and supplementation with propionic acid reduced susceptibility to EAP and corrected the imbalance of Th17/Treg cell differentiation in vivo and in vitro . Furthermore, SCFA receptor G-protein-coupled receptor 43 and intracellular histone deacetylase 6 regulated by propionic acid in Th17 and Treg cells were also evaluated. Lastly, we observed that fecal transplantation from EAP mice induced the decrease of Treg cell frequency in recipient mice. Our data showed that gut dysbiosis contributed to a Th17/Treg differentiation imbalance in EAP via the decrease of metabolite propionic acid and provided valuable immunological groundwork for further intervention in immunologic derangement of CP/CPPS by targeting propionic acid., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Du, Yue, Niu, Liu, Zhang, Chen, Chen, Guan, Hua, Li, Chen, Zhang and Liang.)

Background: Triple negative breast cancer (TNBC) is characterized by poor prognosis and a lack of effective therapeutic agents owing to the absence of biomarkers. A high abundance of tumor-infiltrating regulatory T cells (Tregs) was associated with worse prognosis in malignant disease. Exploring the association between Treg cell infiltration and TNBC will provide new insights for understanding TNBC immunosuppression and may pave the way for developing novel immune-based treatments., Materials and Methods: Patients from TCGA were divided into Treg-high (Treg-H) and Treg-low (Treg-L) groups based on the abundance of Tregs according to CIBERSORT analysis. The association between expression level of Tregs and the clinical characteristics as well as prognosis of breast cancer were evaluated. Next, a Treg-related prognostic model was established after survival-dependent univariate Cox and LASSO regression analysis, companied with an external GEO cohort validation. Then, GO, KEGG and GSEA analyses were performed between the Treg-H and Treg-L groups. Masson and Sirius red/Fast Green staining were applied for ECM characterization. Accordingly, Jurkat T cells were encapsulated in 3D collagen to mimic the ECM microenvironment, and the expression levels of CD4, FOXP3 and CD25 were quantified according to immunofluorescence staining., Results: The expression level of Tregs is significantly associated with the clinical characteristics of breast cancer patients, and a high level of Treg cell expression indicates a poor prognosis in TNBC. To further evaluate this, a Treg-related prognostic model was established that accurately predicted outcomes in both TCGA training and GEO validation cohorts of TNBC patients. Subsequently, ECM-associated signaling pathways were identified between the Treg-H and Treg-L groups, indicating the role of ECM in Treg infiltration. Since we found increasing collagen concentrations in TNBC patients with distant migration, we encapsulated Jurkat T cells within a 3D matrix with different collagen concentrations and observed that increasing collagen concentrations promoted the expression of Treg biomarkers, supporting the regulatory role of ECM in Treg infiltration., Conclusion: Our results support the association between Treg expression and breast cancer progression as well as prognosis in the TNBC subtype. Moreover, increasing collagen density may promote Treg infiltration, and thus induce an immunosuppressed TME., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gao, Tian, Zhou, Zhu, Lu, Ma, Feng, Jiang and Wang.)

CD4 + CD25 + Foxp3 + Regulatory (Treg) T cells are mainly generated within the thymus. However, the mechanism of thymic Treg cell (tTreg cell) generation remains to be fully revealed. Although the functions of TCR/CD28 co-stimulation have been widely accepted, the functions of cytokines in the generation of tTreg cells remain highly controversial. In this review, we summarize the existing studies on cytokine regulation of tTreg cell generation. By integrating the key findings of cytokines in tTreg cell generation, we have concluded that four members of γc family cytokines (IL-2, IL-4, IL-7 and IL-15), transforming growth factor β (TGF-β), and three members of TNF superfamily cytokines (GITRL, OX40L and TNF-α) play vitally important roles in regulating tTreg cell generation. We also point out all disputed points and highlight critical scientific questions that need to be addressed in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tang, Jia, Nan, Zuo, Yuan and Zhang.)

Blood vessels are indispensable for host survival and are protected from inappropriate inflammation by immune privilege. This protection is lost in patients with autoimmune vasculitides, a heterogeneous group of diseases causing damage to arteries, arterioles, and capillaries. Vasculitis leads to vascular wall destruction and/or luminal occlusion, resulting in hemorrhage and tissue ischemia. Failure in the quantity and quality of immunosuppressive regulatory T cells (Treg) has been implicated in the breakdown of the vascular immune privilege. Emerging data suggest that Treg deficiencies are disease-specific, affecting distinct pathways in distinct vasculitides. Mechanistic studies have identified faulty CD8 + Tregs in Giant Cell Arteritis (GCA), a vasculitis of the aorta and the large aortic branch vessels. Specifically, aberrant signaling through the NOTCH4 receptor expressed on CD8 + Treg cells leads to rerouting of intracellular vesicle trafficking and failure in the release of immunosuppressive exosomes, ultimately boosting inflammatory attack to medium and large arteries. In Kawasaki's disease, a medium vessel vasculitis targeting the coronary arteries, aberrant expression of miR-155 and dysregulated STAT5 signaling have been implicated in undermining CD4 + Treg function. Explorations of mechanisms leading to insufficient immunosuppression and uncontrolled vascular inflammation hold the promise to discover novel therapeutic interventions that could potentially restore the immune privilege of blood vessels and pave the way for urgently needed innovations in vasculitis management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jin, Parreau, Warrington, Koster, Berry, Goronzy and Weyand.)

Trace element iron affects T cell biology, but the knowledge about the role of iron in regulating Treg cell expansion is limited. Treg cells play an important role in keeping peripheral T cell tolerance, increasing Treg cell expansion is a promising therapeutic method for SLE. Here we showed that iron deficiency promotes Treg cell expansion by reducing ROS accumulation, improving the disease progression of pristane-induced lupus. Increased oxidative stress inhibits Treg cell differentiation by inducing cell apoptosis. Our data suggest that altering iron metabolism promotes Treg cell expansion by preventing oxidation-induced cell death, which may provide a potential therapeutic strategy for SLE., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gao, Song, Lu, Hu, Zheng, Jia and Zhao.)

As the major hub of metabolic activity and an organelle sequestering pro-apoptogenic intermediates, mitochondria lie at the crossroads of cellular decisions of death and survival. Intracellular calcium is a key regulator of these outcomes with rapid, uncontrolled uptake into mitochondria, activating pro-apoptotic cascades that trigger cell death. Here, we show that calcium uptake and mitochondrial metabolism in murine T-regulatory cells (Tregs) is tuned by Notch1 activity. Based on analysis of Tregs and the HEK cell line, we present evidence that modulation of cellular calcium dynamics underpins Notch1 regulation of mitochondrial homeostasis and consequently anti-apoptotic activity. Targeted siRNA-mediated ablations reveal dependency on molecules controlling calcium release from the endoplasmic reticulum (ER) and the chaperone, glucose-regulated protein 75 (Grp75), the associated protein Voltage Dependent Anion Channel (VDAC)1 and the Mitochondrial Calcium Uniporter (MCU), which together facilitate ER calcium transfer and uptake into the mitochondria. Endogenous Notch1 is detected in immune-complexes with Grp75 and VDAC1. Deficits in mitochondrial oxidative and survival in Notch1 deficient Tregs, were corrected by the expression of recombinant Notch1 intracellular domain, and in part by recombinant Grp75. Thus, the modulation of calcium dynamics and consequently mitochondrial metabolism underlies Treg survival in conditions of nutrient stress. This work positions a key role for Notch1 activity in these outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Saini, Lakshminarayanan, Kundu and Sarin.)

Among CD4+ T-cells, T helper 17 (Th17) cells play a sentinel role in the defense against bacterial/fungal pathogens at mucosal barriers. However, Th17 cells are also highly susceptible to HIV-1 infection and are rapidly depleted from gut mucosal sites, causing an imbalance of the Th17/Treg ratio and impairing cytokines production. Consequently, damage to the gut mucosal barrier leads to an enhanced microbial translocation and systemic inflammation, a hallmark of HIV-1 disease progression. Th17 cells' expression of mucosal homing receptors (CCR6 and α4β7), as well as HIV receptors and co-receptors (CD4, α4β7, CCR5, and CXCR4), contributes to susceptibility to HIV infection. The up-regulation of numerous intracellular factors facilitating HIV production, alongside the downregulation of factors inhibiting HIV, helps to explain the frequency of HIV DNA within Th17 cells. Th17 cells harbor long-lived viral reservoirs in people living with HIV (PLWH) receiving antiretroviral therapy (ART). Moreover, cell longevity and the proliferation of a fraction of Th17 CD4 T cells allow HIV reservoirs to be maintained in ART patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Renault, Veyrenche, Mennechet, Bedin, Routy, Van de Perre, Reynes and Tuaillon.)

Sepsis is the leading cause of death among patients, especially elderly patients, in intensive care units worldwide. In this study, we established a sepsis model using naturally aged rats and injected 5×10 6 umbilical cord-derived MSCs via the tail vein. Each group of rats was analyzed for survival, examined for biochemical parameters, stained for organ histology, and analyzed for the Th cell subpopulation ratio and inflammatory cytokine levels by flow cytometry. Western blotting was performed to detect the activity of the JAK-STAT signaling pathway. We designed the vitro experiments to confirm the regulatory role of MSCs, and verified the possible mechanism using JAK/STAT inhibitors. It was revealed from the experiments that the 72 h survival rate of sepsis rats treated with MSCs was significantly increased, organ damage and inflammatory infiltration were reduced, the levels of organ damage indicators were decreased, the ratios of Th1/Th2 and Th17/Treg in peripheral blood and spleen were significantly decreased, the levels of pro-inflammatory cytokines such as IL-6 were decreased, the levels of anti-inflammatory cytokines such as IL-10 were increased, and the levels of STAT1 and STAT3 phosphorylation were reduced. These results were validated in in vitro experiments. Therefore, this study confirms that MSCs can control the inflammatory response induced by sepsis by regulating Th cells and inflammatory factors, and that this leads to the reduction of tissue damage, protection of organ functions and ultimately the improvement of survival in aged sepsis model rats. Inhibition of the JAK-STAT signaling pathway was surmised that it may be an important mechanism for their action., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Deng, Sun, Zhao, Li, Yang, Yuan, Liu, Qian, Zhou and Kang.)

Antibody-induced complement activation may cause injury of the neuromuscular junction (NMJ) and is thus considered as a primary pathogenic factor in human myasthenia gravis (MG) and animal models of experimental autoimmune myasthenia gravis (EAMG). In this study, we tested whether CRIg/FH, a targeted complement inhibitor, could attenuate NMJ injury in rat MG models. We first demonstrated that CRIg/FH could inhibit complement-dependent cytotoxicity on human rhabdomyosarcoma TE671 cells induced by MG patient-derived IgG in vitro . Furthermore, we investigated the therapeutic effect of CRIg/FH in a passive and an active EAMG rodent model. In both models, administration of CRIg/FH could significantly reduce the complement-mediated end-plate damage and suppress the development of EAMG. In the active EAMG model, we also found that CRIg/FH treatment remarkably reduced the serum concentration of autoantibodies and of the cytokines including IFN-γ, IL-2, IL-6, and IL-17, and upregulated the percentage of Treg cells in the spleen, which was further verified in vitro . Therefore, our findings indicate that CRIg/FH may hold the potential for the treatment of MG via immune modulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Song, Zhao, Yan, Luo, Xi, Ding, Li, Hu and Zhao.)

The oral mucosa is constantly exposed to a plethora of stimuli including food antigens, commensal microbiota and pathogens, requiring distinct immune responses. We previously reported that human oral epithelial cells (OECs) suppress immune responses to bacteria, using H413 and TR146 OEC lines and primary OECs in co-culture with dendritic cells (DCs) and T cells (OEC-conditioned cells). OECs reduced DCs expression of CD80/CD86 and IL-12/TNFα release and impaired T cell activation. Here, we further evaluated the immunosuppression by these OECs and investigated the underlying mechanisms. OEC-conditioned DCs did not induce CD4 T cell polarization towards Treg, judging by the absence of FoxP3 expression. OECs also repressed T-bet/IFNγ expression in CD4 and CD8 T cells activated by DCs or anti-CD3/CD28 antibodies. This inhibition depended on OEC:T cell ratio and IFNγ repression occurred at the transcriptional level. Time-lapse experiments showed that OECs inhibited early steps of T cell activation, consistent with OECs inability to suppress T cells stimulated with PMA/ionomycin. Blocking CD40/CD40L, CD58/CD2 and PD-L1/PD-1 interactions with specific antibodies did not disrupt T cell suppression by OECs. However, preventing prostaglandin E2 (PGE2) synthesis or blocking PGE2 binding to the cognate EP2/EP4 receptors, restored IFNγ and TNFα production in OEC-conditioned T cells. Finally, treating OECs with poly(I:C), which simulates viral infections, limited T cell suppression. Overall, these results point to an inherent ability of OECs to suppress immune responses, which can nonetheless be eluded when OECs are under direct assault., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sanchez-Trincado, Pelaez-Prestel, Lafuente and Reche.)

Treg therapies are being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs remains controversial. We challenged human Tregs ex-vivo with pro-inflammatory cytokines IL-6 and TNF α and observed greatly enhanced proliferation stimulated by anti-CD3 and anti-CD28 (aCD3/28) beads or CD28 superagonist (CD28SA). The cytokine-exposed Tregs maintained high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low IFNγ, IL-4, and IL-17 secretion. Blocking TNF receptor using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression. These results prompted us to consider using CD28SA together with IL-6 and TNF α without aCD3/28 beads (beadless) as an alternative protocol for therapeutic Treg manufacturing. Metabolomics profiling revealed more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential during beadless Treg expansion. Finally, beadless expanded Tregs maintained suppressive functions in vitro and in vivo . These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function. This property can be harnessed for therapeutic Treg manufacturing., Competing Interests: QT is a co-founder and scientific advisor of Sonoma Biotherapeutics. QT, NS, and FV are co-inventors of a patent on manufacturing Tregs based on results from this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Skartsis, Peng, Ferreira, Nguyen, Ronin, Muller, Vincenti and Tang.)

Deoxynivalenol (DON), a highly prevalent contaminant of grain-based products, is known to induce reproductive- and immunotoxicities. Considering the importance of immune development in early life, the present study investigated the effects of perinatal DON exposure on allergy development and vaccine responsiveness in the offspring. Pregnant mice received control or DON-contaminated diets (12.5 mg/kg diet) during pregnancy and lactation. After weaning, female offspring were sensitized to ovalbumin (OVA) by oral administration of OVA with cholera toxin (CT). Male offspring were injected with Influvac vaccine. OVA-specific acute allergic skin response (ASR) in females and vaccine-specific delayed-type hypersensitivity (DTH) in males were measured upon intradermal antigen challenge. Immune cell populations in spleen and antigen-specific plasma immunoglobulins were analyzed. In female CT+OVA-sensitized offspring of DON-exposed mothers ASR and OVA-specific plasma immunoglobulins were significantly higher, compared to the female offspring of control mothers. In vaccinated male offspring of DON-exposed mothers DTH and vaccine-specific antibody levels were significantly lower, compared to the male offspring of control mothers. In both models a significant reduction in regulatory T cells, Tbet + Th1 cells and Th1-related cytokine production of the offspring of DON-exposed mothers was observed. In conclusion, early life dietary exposure to DON can adversely influence immune development in the offspring. Consequently, the immune system of the offspring may be skewed towards an imbalanced state, resulting in an increased allergic immune response to food allergens and a decreased immune response to vaccination against influenza virus in these models., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Seyed Toutounchi, Braber, van’t Land, Thijssen, Garssen, Kraneveld, Folkerts and Hogenkamp.)

This study was initiated to better understand the nature of innate immune responses and the relatively weak and delayed immune response against porcine reproductive and respiratory syndrome virus (PRRSV). Following modified live virus (MLV) vaccination or infection with two PRRSV-2 strains, we analyzed the transcriptome of peripheral blood mononuclear cells collected before and at three and seven days after vaccination or infection. We used blood transcriptional modules (BTMs)-based gene set enrichment analyses. BTMs related to innate immune processes were upregulated by PRRSV-2 strains but downregulated by MLV. In contrast, BTMs related to adaptive immune responses, in particular T cells and cell cycle, were downregulated by PRRSV-2 but upregulated by MLV. In addition, we found differences between the PRRSV strains. Only the more virulent strain induced a strong platelet activation, dendritic cell activation, interferon type I and plasma cell responses. We also calculated the correlations of BTM with the neutralizing antibody and the T-cell responses. Early downregulation (day 0-3) of dendritic cell and B-cell BTM correlated to both CD4 and CD8 T-cell responses. Furthermore, a late (day 3-7) upregulation of interferon type I modules strongly correlated to helper and regulatory T-cell responses, while inflammatory BTM upregulation correlated more to CD8 T-cell responses. BTM related to T cells had positive correlations at three days but negative associations at seven days post-infection. Taken together, this work contributes to resolve the complexity of the innate and adaptive immune responses against PRRSV and indicates a fundamentally different immune response to the less immunogenic MLV compared to field strains which induced robust adaptive immune responses. The identified correlates of T-cell responses will facilitate a rational approach to improve the immunogenicity of MLV., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bocard, Kick, Hug, Lischer, Käser and Summerfield.)