Type 1 regulatory (Tr1) T cells are a major source of IL-10-mediated immune regulation, yet their phenotypic definition and role in human disease remain incompletely understood. In this issue of the Journal of Clinical Investigation, Nideffer et al. provide insight into human Tr1 cells during pediatric Plasmodium falciparum (Pf) infection. The authors identified Tr1 cells as a major component of the malaria-specific CD4+ T cell response, producing both IL-10 and IFN-γ. They proposed that, in this context, Tr1 cells may be better identified by CD127 downregulation combined with CXCR6 expression than by other surface markers. Importantly, Tr1 cells exhibited suppressive function and were associated with reduced symptomatic disease but also with prolonged infection. Together, these findings refine current models of Tr1 cell identity and establish a more rigorous framework for marker validation using single-cell transcriptomics while highlighting the role of Tr1 cells in balancing immunity and immunopathology during infection.

Targeted gene manipulation in a complex microbial community is an enabling technology for precise microbiome editing. Here we introduce BACTRINS, an in situ microbiome engineering platform designed for efficient and precise genomic insertion of a desired payload and simultaneous knockout of target genes. This system leverages conjugation-mediated delivery of CRISPR-associated transposases to achieve RNA-guided genomic integration, allowing precise insertion of a therapeutic payload while neutralizing pathogen virulence without causing cell death. When applied against an Enterobacteriaceae Shiga toxin-producing pathogen in the gut, this system delivers a CRISPR-associated transposase by bacterial conjugation for site-specific inactivation of the Shiga toxin gene and integration of a nanobody therapeutic payload to disrupt pathogen attachment. A single dose of this therapy results in high-efficiency Shiga gene inactivation and improved survival in a murine infection model of Shiga-producing pathogen. This work establishes a new type of live bacterial therapeutic capable of reducing gut infections by transforming toxigenic pathogens into commensal protectors. A self-transmissible CRISPR-associated transposase system encodes a nanobody payload to treat Shiga toxin infections.

Barrier organs such as the gastrointestinal tract, lungs, and skin are colonized by diverse microbial strains, including bacteria, viruses, and fungi. These microorganisms, collectively known as the commensal microbiota, play critical roles in maintaining health by defending against pathogens, metabolizing nutrients, and providing essential metabolites. In the gut, commensal-derived antigens are frequently sensed by the intestinal immune system. Maintaining tolerance toward these beneficial microbial species is crucial, as failure to do so can lead to chronic inflammatory conditions like inflammatory bowel disease (IBD) and can even affect systemic immune or metabolic health. The immune system carefully regulates responses to commensals through various mechanisms, including the induction of anti-inflammatory CD4⁺ T cell responses. Foxp3⁺ regulatory T cells (Foxp3+ Tregs) and Type 1 regulatory T cells (Tr1) play a major role in promoting tolerance, as both cell types can produce the anti-inflammatory cytokine IL-10. In addition to these regulatory T cells, effector T cell subsets, such as Th17 cells, also adopt anti-inflammatory functions within the intestine in response to the microbiota. This process of anti-inflammatory CD4+ T cell induction is heavily influenced by the microbiota and their metabolites. Microbial metabolites affect intestinal epithelial cells, promoting the secretion of anti-inflammatory mediators that create a tolerogenic environment. They also modulate intestinal dendritic cells (DCs) and macrophages, inducing a tolerogenic state, and can interact directly with T cells to drive anti-inflammatory CD4⁺ T cell functionality. The disrupted balance of these signals may result in chronic inflammation, with broader implications for systemic health. In this review, we highlight the intricate interplays between commensal microorganisms and the immune system in the gut. We discuss how the microbiota influences the differentiation of commensal-specific anti-inflammatory CD4⁺ T cells, such as Foxp3⁺ Tregs, Tr1 cells, and Th17 cells, and explore the mechanisms through which microbial metabolites modulate these processes. We further discuss the innate signals that prime and commit these cells to an anti-inflammatory fate.

Western diets reduced in fiber promote dysbiosis and exacerbate colitis, with short-chain fatty acids (SCFAs) from fiber fermentation known to regulate glucagon-like peptide 1 (GLP-1) secretion. Whether GLP-1 dysregulation directly links diet-induced dysbiosis to colitis severity, and if this pathway can be therapeutically targeted independently of dietary fiber, remains unclear. Here, we show that dextran sulfate sodium (DSS)-induced colitis severity correlates with compensatory GLP-1 increases, while receptor blockade worsens damage, confirming GLP-1's protective role during colitis. Fiber deficiency impaired L cell function and GLP-1 release, increasing colitis susceptibility. GLP-1 receptor agonist reversed these effects, restoring barrier integrity and accelerating recovery. We engineered a probiotic, releasing a microbial peptide, that locally elevates GLP-1, which normalized gut parameters in fiber-deprived mice and alleviated colitis via GLP-1-dependent mechanisms, including improved metabolism, antimicrobial defenses , and barrier restoration. Our findings mechanistically connect fiber deficiency to colitis through GLP-1 and demonstrate that probiotic-mediated GLP-1 modulation can bypass dietary fiber requirements to maintain gut homeostasis.

Commensal microbes induce cytokine-producing effector tissue-resident CD4+ T cells, but the function of these T cells in mucosal homeostasis is not well understood. Here, we report that commensal-specific intestinal Th17 cells possess an anti-inflammatory phenotype marked by expression of interleukin (IL)-10 and co-inhibitory receptors. The anti-inflammatory phenotype of gut-resident commensal-specific Th17 cells was driven by the transcription factor c-MAF. IL-10-producing commensal-specific Th17 cells were heterogeneous and derived from a TCF1+ gut-resident progenitor Th17 cell population. Th17 cells acquired IL-10 expression and anti-inflammatory phenotype in the small-intestinal lamina propria. IL-10 production by CD4+ T cells and IL-10 signaling in intestinal macrophages drove IL-10 expression by commensal-specific Th17 cells. Intestinal commensal-specific Th17 cells possessed immunoregulatory functions and curbed effector T cell activity in vitro and in vivo in an IL-10-dependent and c-MAF-dependent manner. Our results suggest that tissue-resident commensal-specific Th17 cells perform regulatory functions in mucosal homeostasis.

A major challenge facing tumor-antigen targeting therapies such as chimeric antigen receptor (CAR)–T cells is the identification of suitable targets that are specifically and uniformly expressed on heterogeneous solid tumors. By contrast, certain species of bacteria selectively colonize immune-privileged tumor cores and can be engineered as antigen-independent platforms for therapeutic delivery. To bridge these approaches, we developed a platform of probiotic-guided CAR-T cells (ProCARs), in which tumor-colonizing probiotics release synthetic targets that label tumor tissue for CAR-mediated lysis in situ. This system demonstrated CAR-T cell activation and antigen-agnostic cell lysis that was safe and effective in multiple xenograft and syngeneic models of human and mouse cancers. We further engineered multifunctional probiotics that co-release chemokines to enhance CAR-T cell recruitment and therapeutic response.

BACKGROUND & AIMS:The liver is one of the organs most commonly affected by metastasis. The presence of liver metastases has been reported to be responsible for an immunosuppressive microenvironment and diminished immunotherapy efficacy. Herein, we aimed to investigate the role of IL-10 in liver metastasis and to determine how its modulation could affect the efficacy of immunotherapy in vivo. METHODS:To induce spontaneous or forced liver metastasis in mice, murine cancer cells (MC38) or colon tumor organoids were injected into the cecum or the spleen, respectively. Mice with complete and cell type-specific deletion of IL-10 and IL-10 receptor alpha were used to identify the source and the target of IL-10 during metastasis formation. Programmed death ligand 1 (PD-L1)-deficient mice were used to test the role of this checkpoint. Flow cytometry was applied to characterize the regulation of PD-L1 by IL-10. RESULTS:We found that Il10-deficient mice and mice treated with IL-10 receptor alpha antibodies were protected against liver metastasis formation. Furthermore, by using IL-10 reporter mice, we demonstrated that Foxp3+ regulatory T cells (Tregs) were the major cellular source of IL-10 in liver metastatic sites. Accordingly, deletion of IL-10 in Tregs, but not in myeloid cells, led to reduced liver metastasis. Mechanistically, IL-10 acted on Tregs in an autocrine manner, thereby further amplifying IL-10 production. Furthermore, IL-10 acted on myeloid cells, i.e. monocytes, and induced the upregulation of the immune checkpoint protein PD-L1. Finally, the PD-L1/PD-1 axis attenuated CD8-dependent cytotoxicity against metastatic lesions. CONCLUSIONS:Treg-derived IL-10 upregulates PD-L1 expression in monocytes, which in turn reduces CD8+ T-cell infiltration and related antitumor immunity in the context of colorectal cancer-derived liver metastases. These findings provide the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastases. IMPACT AND IMPLICATIONS:Liver metastasis diminishes the effectiveness of immunotherapy and increases the mortality rate in patients with colorectal cancer. We investigated the role of IL-10 in liver metastasis formation and assessed its impact on the effectiveness of immunotherapy. Our data show that IL-10 is a pro-metastatic factor involved in liver metastasis formation and that it acts as a regulator of PD-L1. This provides the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastasis.

During metastasis, cancer cells invade, intravasate, enter the circulation, extravasate, and colonize target organs. Here, we examined the role of interleukin (IL)-22 in metastasis. Immune cell-derived IL-22 acts on epithelial tissues, promoting regeneration and healing upon tissue damage, but it is also associated with malignancy. Il22-deficient mice and mice treated with an IL-22 antibody were protected from colon-cancer-derived liver and lung metastasis formation, while overexpression of IL-22 promoted metastasis. Mechanistically, IL-22 acted on endothelial cells, promoting endothelial permeability and cancer cell transmigration via induction of endothelial aminopeptidase N. Multi-parameter flow cytometry and single-cell sequencing of immune cells isolated during cancer cell extravasation into the liver revealed iNKT17 cells as source of IL-22. iNKT-cell-deficient mice exhibited reduced metastases, which was reversed by injection of wild type, but not Il22-deficient, invariant natural killer T (iNKT) cells. IL-22-producing iNKT cells promoting metastasis were tissue resident, as demonstrated by parabiosis. Thus, IL-22 may present a therapeutic target for prevention of metastasis.

Hepatocellular carcinoma (HCC) ranks among the five most common cancer entities worldwide and leads to hundred-thousands of deaths every year. Despite some groundbreaking therapeutical revelations during the last years, the overall prognosis remains poor. Although the immune system fights malignant transformations with a robust anti-tumor response, certain immune mediators have also been shown to promote cancer development. For example, interleukin (IL)-22 has been associated with HCC progression and worsened prognosis in multiple studies. However, the underlying mechanisms of the pathological role of IL-22-signaling as well as the role of its natural antagonist IL-22 binding protein (IL-22BP) in HCC remain elusive. Here, we corroborate the pathogenic role of IL-22 in HCC by taking advantage of two mouse models. Moreover, we observed a protective role of IL-22BP during liver carcinogenesis. While IL-22 was mainly produced by CD4+ T cells in HCC, IL-22BP was abundantly expressed by neutrophils during liver carcinogenesis. Hepatocytes could be identified as a major target of this pathological IL-22-signaling. Moreover, abrogation of IL-22 signaling in hepatocytes in IL22ra1flox/flox × AlbCre+ mice reduced STEAP4 expression-a known oncogene-in HCC in vivo. Likewise, STEAP4 expression correlated with IL22 levels in human HCC samples, but not in healthy liver specimens. In conclusion, these data encourage the development of therapeutical approaches that target the IL-22–IL-22BP axis in HCC.

Interleukin (IL)-10 is considered a prototypical anti-inflammatory cytokine, significantly contributing to the maintenance and reestablishment of immune homeostasis. Accordingly, it has been shown in the intestine that IL-10 produced by Tregs can act on effector T cells, thereby limiting inflammation. Herein, we investigate whether this role also applies to IL-10 produced by T cells during central nervous system (CNS) inflammation. During neuroinflammation, both CNS-resident and -infiltrating cells produce IL-10; yet, as IL-10 has a pleotropic function, the exact contribution of the different cellular sources is not fully understood. We find that T-cell-derived IL-10, but not other relevant IL-10 sources, can promote inflammation in experimental autoimmune encephalomyelitis. Furthermore, in the CNS, T-cell-derived IL-10 acts on effector T cells, promoting their survival and thereby enhancing inflammation and CNS autoimmunity. Our data indicate a pro-inflammatory role of T-cell-derived IL-10 in the CNS.

Abstract Background Lymphocytes have dichotomous functions in ischemic stroke. Regulatory T cells are protective, while IL-17A from innate lymphocytes promotes the infarct growth. With recent advances of T cell-subtype specific transgenic mouse models it now has become possible to study the complex interplay of T cell subpopulations in ischemic stroke. Methods In a murine model of experimental stroke we analyzed the effects of IL-10 on the functional outcome for up to 14 days post-ischemia and defined the source of IL-10 in ischemic brains based on immunohistochemistry, flow cytometry, and bone-marrow chimeric mice. We used neutralizing IL-17A antibodies, intrathecal IL-10 injections, and transgenic mouse models which harbor a deletion of the IL-10R on distinct T cell subpopulations to further explore the interplay between IL-10 and IL-17A pathways in the ischemic brain. Results We demonstrate that IL-10 deficient mice exhibit significantly increased infarct sizes on days 3 and 7 and enlarged brain atrophy and impaired neurological outcome on day 14 following tMCAO. In ischemic brains IL-10 producing immune cells included regulatory T cells, macrophages, and microglia. Neutralization of IL-17A following stroke reversed the worse outcome in IL-10 deficient mice and intracerebral treatment with recombinant IL-10 revealed that IL-10 controlled IL-17A positive lymphocytes in ischemic brains. Importantly, IL-10 acted differentially on αβ and γδ T cells. IL-17A producing CD4+ αβ T cells were directly controlled via their IL-10-receptor (IL-10R), whereas IL-10 by itself had no direct effect on the IL-17A production in γδ T cells. The control of the IL-17A production in γδ T cells depended on an intact IL10R signaling in regulatory T cells (Tregs). Conclusions Taken together, our data indicate a key function of IL-10 in restricting the detrimental IL-17A-signaling in stroke and further supports that IL-17A is a therapeutic opportunity for stroke treatment.

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background & Aims Unregulated activity of interleukin (IL) 22 promotes intestinal tumorigenesis in mice. IL22 binds the antagonist IL22 subunit alpha 2 (IL22RA2, also called IL22BP). We studied whether alterations in IL22BP contribute to colorectal carcinogenesis in humans and mice. Methods We obtained tumor and nontumor tissues from patients with colorectal cancer (CRC) and measured levels of cytokines by quantitative polymerase chain reaction, flow cytometry, and immunohistochemistry. We measured levels of Il22bp messenger RNA in colon tissues from wild-type, Tnf–/–, Lta–/–, and Ltb–/– mice. Mice were given azoxymethane and dextran sodium sulfate to induce colitis and associated cancer or intracecal injections of MC38 tumor cells. Some mice were given inhibitors of lymphotoxin beta receptor (LTBR). Intestine tissues were analyzed by single-cell sequencing to identify cell sources of lymphotoxin. We performed immunohistochemistry analysis of colon tissue microarrays from patients with CRC (1475 tissue cores, contained tumor and nontumor tissues) and correlated levels of IL22BP with patient survival times. Results Levels of IL22BP were decreased in human colorectal tumors, compared with nontumor tissues, and correlated with levels of lymphotoxin. LTBR signaling was required for expression of IL22BP in colon tissues of mice. Wild-type mice given LTBR inhibitors had an increased tumor burden in both models, but LTBR inhibitors did not increase tumor growth in Il22bp–/– mice. Lymphotoxin directly induced expression of IL22BP in cultured human monocyte–derived dendritic cells via activation of nuclear factor κB. Reduced levels of IL22BP in colorectal tumor tissues were associated with shorter survival times of patients with CRC. Conclusions Lymphotoxin signaling regulates expression of IL22BP in colon; levels of IL22BP are reduced in human colorectal tumors, associated with shorter survival times. LTBR signaling regulates expression of IL22BP in colon tumors in mice and cultured human dendritic cells. Patients with colorectal tumors that express low levels of IL22BP might benefit from treatment with an IL22 antagonist.

IL-10 is a prototypical anti-inflammatory cytokine, which is fundamental to the maintenance of immune homeostasis, especially in the intestine. There is an assumption that cells producing IL-10 have an immunoregulatory function. However, here we report that IL-10-producing CD4 + T cells are phenotypically and functionally heterogeneous. By combining single cell transcriptome and functional analyses, we identified a subpopulation of IL-10-producing Foxp3 neg CD4 + T cells that displays regulatory activity unlike other IL-10-producing CD4 + T cells, which are unexpectedly pro-inflammatory. The combinatorial expression of co-inhibitory receptors is sufficient to discriminate IL-10-producing CD4 + T cells with regulatory function from others and to identify them across different tissues and disease models in mice and humans. These regulatory IL-10-producing Foxp3 neg CD4 + T cells have a unique transcriptional program, which goes beyond the regulation of IL-10 expression. Finally, we found that patients with Inflammatory Bowel Disease demonstrate a deficiency in this specific regulatory T-cell subpopulation.

Inflammatory bowel disease (IBD) is caused by the interplay of various factors. It occurs in genetically susceptible people due to dysregulated immune responses to several unknown antigens, including those derived from the commensal microbiota. Effector T-helper cells, especially TH17 cells, are considered a major driver of disease progression. The endogenous resident counterparts of effector T-helper cells are the regulatory T cells, mainly Foxp3+ Treg cells and type 1 regulatory (TR1) T cells. Both have strong immune regulatory capacity and can terminate immune responses. Interestingly, the expression of IL-10 receptor on regulatory T cells has a high impact on the regulatory capacity of these cells. Inflammatory bowel disease is becoming a global health issue. No curative therapy is currently available. However, initial clinical trials have been conducted successfully, proving the safety of a regulatory T-cell-based therapy. This therapy might lead to long-lasting remission and to a possible cure for IBD. This review provides a summary of the current findings and the outcome of the clinical trials based on T-cell therapy for IBD and for other inflammatory conditions.

A common helper-like innate lymphoid precursor (CHILP) restricted to the innate lymphoid cells (ILC) lineage has been recently characterized. While specific requirements of transcription factors for CHILPs development has been partially described, their ability to sense cytokines and react to peripheral inflammation remains unaddressed. Here, we found that systemic increase in Flt3L levels correlated with the expansion of Lineage (Lin) neg α4β7 + precursors in the adult murine bone marrow. Expanded Lin neg α4β7 + precursors were bona fide CHILPs as seen by their ability to differentiate into all helper ILCs subsets but cNK in vivo . Interestingly, Flt3L-expanded CHILPs transferred into lymphopenic mice preferentially reconstituted the small intestine. While we did not observe changes in serum Flt3L during DSS-induced colitis in mice or plasma from inflammatory bowel disease (IBD) patients, elevated Flt3L levels were detected in acute malaria patients. Interestingly, while CHILP numbers were stable during the course of DSS-induced colitis, they expanded following increased serum Flt3L levels in malaria-infected mice, hence suggesting a role of the Flt3L-ILC axis in malaria. Collectively, our results indicate that Flt3L expands CHILPs in the bone marrow, which might be associated with specific inflammatory conditions.

Wound healing is a crucial process which protects our body against permanent damage and invasive infectious agents. Upon tissue damage, inflammation is an early event which is orchestrated by a multitude of innate and adaptive immune cell subsets including TH17 cells. TH17 cells and TH17 cell associated cytokines can impact wound healing positively by clearing pathogens and modulating mucosal surfaces and epithelial cells. Injury of the gut mucosa can cause fast expansion of TH17 cells and their induction from naïve T cells through Interleukin (IL)-6, TGF-β, and IL-1β signaling. TH17 cells produce various cytokines, such as tumor necrosis factor (TNF)-α, IL-17, and IL-22, which can promote cell survival and proliferation and thus tissue regeneration in several organs including the skin, the intestine, and the liver. However, TH17 cells are also potentially pathogenic if not tightly controlled. Failure of these control mechanisms can result in chronic inflammatory conditions, such as Inflammatory Bowel Disease (IBD), and can ultimately promote carcinogenesis. Therefore, there are several mechanisms which control TH17 cells. One control mechanism is the regulation of TH17 cells via regulatory T cells and IL-10. This mechanism is especially important in the intestine to terminate immune responses and maintain homeostasis. Furthermore, TH17 cells have the potential to convert from a pro-inflammatory phenotype to an anti-inflammatory phenotype by changing their cytokine profile and acquiring IL-10 production, thereby limiting their own pathological potential. Finally, IL-22, a signature cytokine of TH17 cells, can be controlled by an endogenous soluble inhibitory receptor, Interleukin 22 binding protein (IL-22BP). During tissue injury, the production of IL-22 by TH17 cells is upregulated in order to promote tissue regeneration. To limit the regenerative program, which could promote carcinogenesis, IL-22BP is upregulated during the later phase of regeneration in order to terminate the effects of IL-22. This delicate balance secures the beneficial effects of IL-22 and prevents its potential pathogenicity. An important future goal is to understand the precise mechanisms underlying the regulation of TH17 cells during inflammation, wound healing, and carcinogenesis in order to design targeted therapies for a variety of diseases including infections, cancer, and immune mediated inflammatory disease.

The intestine is colonized by hundreds of different species of commensal bacteria, viruses, and fungi. Therefore, the intestinal immune system is constantly being challenged by foreign antigens. The immune system, the commensal microbiota, and the intestinal epithelial surface have to maintain a tight balance to guarantee defense against potential pathogens and to prevent chronic inflammatory conditions at the same time. Failure of these mechanisms can lead to a vicious cycle in which a perpetual tissue damage/repair process results in a pathological reorganization of the normal mucosal surface. This dysregulation of the intestine is considered to be one of the underlying causes for both inflammatory bowel disease (IBD) and colorectal cancer. TH17 cells have been associated with immune-mediated diseases, such as IBD, since their discovery in 2005. Upon mucosal damage, these cells are induced by a combination of different cytokines, such as IL-6, TGF-β, and IL-1β. TH17 cells are crucial players in the defense against extracellular pathogens and have various mechanisms to fulfill their function. They can activate and attract phagocytic cells. Additionally, TH17 cells can induce the release of anti-microbial peptides from non-immune cells, such as epithelial cells. The flip side of the coin is the strong potential of TH17 cells to be pro-inflammatory and promote pathogenicity. TH17 cells have been linked to both mucosal regeneration and inflammation. In turn, these cells and their cytokines emerged as potential therapeutic targets both for inflammatory diseases and cancer. This review will summarize the current knowledge regarding the TH17 cell-enterocyte crosstalk and give an overview of its clinical implications.