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Chemotherapy offers long-term clinical benefits to many cancer patients. However, several pre-clinical studies have demonstrated that certain cytotoxic drugs enhance metastasis via multiple mechanisms. These studies have mainly focused on tumor cell-derived inflammation. The importance of host responses triggered by chemotherapy in regulating cancer metastasis has not been fully explored. Here, we showed that multi-dose Gemcitabine (GEM) treatment promoted breast cancer lung metastasis in a transgenic spontaneous breast cancer animal model. Both CCR2+macrophages and monocytes were increased in the lungs of GEM-treated mice. Further, the increase of CCR2+macrophages and monocytes were observed in naïve (tumor-free) mice after GEM treatment. These changes were largely caused by chemotherapy-induced reactive myelopoiesis that are biased toward monocyte development. Mechanistically, enhanced production of mitochondrial ROS (mtROS) was observed in GEM-treated BM LSK cells and monocytes. Treatment with the mitochondrial targeted antioxidant abrogated GEM induced hyper differentiation of BM progenitors. In addition, GEM treatment induced up-regulation of host cell-derived CCL2, and CCL2/CCR2 axis played essential role in the pro-metastatic host response induced by chemotherapy. Further, GEM and Paclitaxel (PTX) in combination with Doxorubicin (DOX) treatment resulted in up-regulation of coagulation factor X (FX) in lung interstitial macrophages. Targeting activated FX (FXa) using FXa inhibitor or F10 gene knockdown reduced pro-metastatic effect of chemotherapy-triggered host response. Together, these studies suggest a novel mechanism for chemotherapy induced metastasis via the host response-induced accumulation of monocytes/macrophages and interplay between coagulation and inflammation in the lungs.

BackgroundPancreatic cancer (PC) is a challenging diagnosis that is yet to benefit from the advancements in immuno-oncologic treatments. Irreversible electroporation (IRE), a non-thermal method of tumor ablation, is used in treatment of select patients with locally-advanced unresectable PC and has potentiated the effect of certain immunotherapies. Yeast-derived particulate β-glucan induces trained innate immunity and successfully reduces murine PC tumor burden. This study tests the hypothesis that IRE may augment β-glucan induced trained immunity in the treatment of PC.Methodsβ-Glucan-trained pancreatic myeloid cells were evaluated ex vivo for trained responses and antitumor function after exposure to ablated and unablated tumor-conditioned media. β-Glucan and IRE combination therapy was tested in an orthotopic murine PC model in wild-type and Rag−/−mice. Tumor immune phenotypes were assessed by flow cytometry. Effect of oral β-glucan in the murine pancreas was evaluated and used in combination with IRE to treat PC. The peripheral blood of patients with PC taking oral β-glucan after IRE was evaluated by mass cytometry.ResultsIRE-ablated tumor cells elicited a potent trained response ex vivo and augmented antitumor functionality. In vivo, β-glucan in combination with IRE reduced local and distant tumor burden prolonging survival in a murine orthotopic PC model. This combination augmented immune cell infiltration to the PC tumor microenvironment and potentiated the trained response from tumor-infiltrating myeloid cells. The antitumor effect of this dual therapy occurred independent of the adaptive immune response. Further, orally administered β-glucan was identified as an alternative route to induce trained immunity in the murine pancreas and prolonged PC survival in combination with IRE. β-Glucan in vitro treatment also induced trained immunity in peripheral blood monocytes obtained from patients with treatment-naïve PC. Finally, orally administered β-glucan was found to significantly alter the innate cell landscape within the peripheral blood of five patients with stage III locally-advanced PC who had undergone IRE.ConclusionsThese data highlight a relevant and novel application of trained immunity within the setting of surgical ablation that may stand to benefit patients with PC.

Cellular metabolism has been proposed to govern distinct γδ T cell effector functions, but the underlying molecular mechanisms remain unclear. We show that interleukin-17 (IL-17)–producing γδ T (γδT17) and interferon-γ (IFN-γ)–producing γδ T (γδT1) cells have differential metabolic requirements and that the rate-limiting enzyme isocitrate dehydrogenase 2 (IDH2) acts as a metabolic checkpoint for their effector functions. Intriguingly, the transcription factor c-Maf regulates γδT17 effector function through direct regulation of IDH2 promoter activity. Moreover, mTORC2 affects the expression of c-Maf and IDH2 and subsequent IL-17 production in γδ T cells. Deletion of c-Maf in γδ T cells reduces metastatic lung cancer development, suggesting c-Maf as a potential target for cancer immune therapy. We show that c-Maf also controls IL-17 production in human γδ T cells from peripheral blood and in oral cancers. These results demonstrate a critical role of the transcription factor c-Maf in regulating γδT17 effector function through IDH2-mediated metabolic reprogramming.

Precast beam–column connections, which are difficult to prefabricate and assemble, are critical members of concrete frames in high seismic regions. In this study, a novel type of precast hybrid con...

Macrophages have been linked to tumor initiation, progression, metastasis, and treatment resistance. However, the transcriptional regulation of macrophages driving the protumor function remains elusive. Here, we demonstrate that the transcription factor c-Maf is a critical controller for immunosuppressive macrophage polarization and function in cancer. c-Maf controls many M2-related genes and has direct binding sites within a conserved noncoding sequence of the Csf-1r gene and promotes M2-like macrophage–mediated T cell suppression and tumor progression. c-Maf also serves as a metabolic checkpoint regulating the TCA cycle and UDP-GlcNAc biosynthesis, thus promoting M2-like macrophage polarization and activation. Additionally, c-Maf is highly expressed in tumor-associated macrophages (TAMs) and regulates TAM immunosuppressive function. Deletion of c-Maf specifically in myeloid cells results in reduced tumor burden with enhanced antitumor T cell immunity. Inhibition of c-Maf partly overcomes resistance to anti–PD-1 therapy in a subcutaneous LLC tumor model. Similarly, c-Maf is expressed in human M2 and tumor-infiltrating macrophages/monocytes as well as circulating monocytes of human non–small cell lung carcinoma (NSCLC) patients and critically regulates their immunosuppressive activity. The natural compound β-glucan downregulates c-Maf expression on macrophages, leading to enhanced antitumor immunity in mice. These findings establish a paradigm for immunosuppressive macrophage polarization and transcriptional regulation by c-Maf and suggest that c-Maf is a potential target for effective tumor immunotherapy.

Metastasis is the leading cause of cancer-related deaths and myeloid cells are critical in the metastatic microenvironment. Here, we explore the implications of reprogramming pre-metastatic niche myeloid cells by inducing trained immunity with whole beta-glucan particle (WGP). WGP-trained macrophages had increased responsiveness not only to lipopolysaccharide but also to tumor-derived factors. WGP in vivo treatment led to a trained immunity phenotype in lung interstitial macrophages, resulting in inhibition of tumor metastasis and survival prolongation in multiple mouse models of metastasis. WGP-induced trained immunity is mediated by the metabolite sphingosine-1-phosphate. Adoptive transfer of WGP-trained bone marrow-derived macrophages reduced tumor lung metastasis. Blockade of sphingosine-1-phosphate synthesis and mitochondrial fission abrogated WGP-induced trained immunity and its inhibition of lung metastases. WGP also induced trained immunity in human monocytes, resulting in antitumor activity. Our study identifies the metabolic sphingolipid-mitochondrial fission pathway for WGP-induced trained immunity and control over metastasis.

Innate immune cells detect pathogens through simultaneous stimulation of multiple receptors, but how cells use the receptor crosstalk to elicit context-appropriate responses is unclear. Here, we reveal that the inflammatory response of macrophages from FcγR-TLR2/1 crosstalk inversely depends on the ligand mobility within a model pathogen membrane. The mechanism is that FcγR and TLR2/1 form separate nanoclusters that interact at their interfaces during crosstalk. Less mobile ligands induce stronger interactions and more overlap between the receptor nanoclusters, leading to enhanced signaling. Different from the prevailing view that immune receptors colocalize to synergize their signaling, our results show that FcγR-TLR2/1 crosstalk occurs through interface interactions between non-colocalizing receptor nanoclusters, which are modulated by ligand mobility. This suggests a mechanism by which innate immune cells could use physical properties of ligands to fine-tune host responses.

Our previous work has revealed the ability of CD11b to regulate BCR signaling and control autoimmune disease in mice. However, how CD11b regulates the immune response under normal conditions remains unknown. Through the use of a CD11b knockout model on a nonautoimmune background, we demonstrated that CD11b-deficient mice have an elevated Ag-specific humoral response on immunization. Deletion of CD11b resulted in elevated low-affinity and high-affinity IgG Ab and increases in Ag-specific germinal center B cells and plasma cells (PCs). Examination of BCR signaling in CD11b-deficient mice revealed defects in association of negative regulators pLyn and CD22 with the BCR, but increases in colocalizations between positive regulator pSyk and BCR after stimulation. Using a CD11b-reporter mouse model, we identified multiple novel CD11b-expressing B cell subsets that are dynamically altered during immunization. Subsequent experiments using a cell-specific CD11b deletion model revealed this effect to be B cell intrinsic and not altered by myeloid cell CD11b expression. Importantly, CD11b expression on PCs also impacts on BCR repertoire selection and diversity in autoimmunity. These studies describe a novel role for CD11b in regulation of the healthy humoral response and autoimmunity, and reveal previously unknown populations of CD11b-expressing B cell subsets, suggesting a complex function for CD11b in B cells during development and activation.

SARS coronavirus 2 (SARS-CoV-2) is a novel viral pathogen that causes a clinical disease called coronavirus disease 2019 (COVID-19). Although most COVID-19 cases are asymptomatic or involve mild upper respiratory tract symptoms, a significant number of patients develop severe or critical disease. Patients with severe COVID-19 commonly present with viral pneumonia that may progress to life-threatening acute respiratory distress syndrome (ARDS). Patients with COVID-19 are also predisposed to venous and arterial thromboses that are associated with a poorer prognosis. The present study identified the emergence of a low-density inflammatory neutrophil (LDN) population expressing intermediate levels of CD16 (CD16Int) in patients with COVID-19. These cells demonstrated proinflammatory gene signatures, activated platelets, spontaneously formed neutrophil extracellular traps, and enhanced phagocytic capacity and cytokine production. Strikingly, CD16Int neutrophils were also the major immune cells within the bronchoalveolar lavage fluid, exhibiting increased CXCR3 but loss of CD44 and CD38 expression. The percentage of circulating CD16Int LDNs was associated with D-dimer, ferritin, and systemic IL-6 and TNF-α levels and changed over time with altered disease status. Our data suggest that the CD16Int LDN subset contributes to COVID-19–associated coagulopathy, systemic inflammation, and ARDS. The frequency of that LDN subset in the circulation could serve as an adjunct clinical marker to monitor disease status and progression.

Despite the remarkable success of immunotherapy in many types of cancer, pancreatic ductal adenocarcinoma has yet to benefit. Innate immune cells are critical to anti-tumor immunosurveillance and recent studies have revealed that these populations possess a form of memory, termed trained innate immunity, which occurs through transcriptomic, epigenetic, and metabolic reprograming. Here we demonstrate that yeast-derived particulate β-glucan, an inducer of trained immunity, traffics to the pancreas, which causes a CCR2-dependent influx of monocytes/macrophages to the pancreas that display features of trained immunity. These cells can be activated upon exposure to tumor cells and tumor-derived factors, and show enhanced cytotoxicity against pancreatic tumor cells. In orthotopic models of pancreatic ductal adenocarcinoma, β-glucan treated mice show significantly reduced tumor burden and prolonged survival, which is further enhanced when combined with immunotherapy. These findings characterize the dynamic mechanisms and localization of peripheral trained immunity and identify an application of trained immunity to cancer.

Lung metastasis-associated macrophages (MAMs) play a very important role in tumor metastasis. In mouse models of lung metastasis, interstitial macrophages (IMs) markedly accumulate and differentiate into MAMs. Several pre-clinical studies have demonstrated that certain cytotoxic drugs enhance metastasis. In this study, we hypothesize that host responses to chemotherapy treatment enhance breast cancer metastasis by modulation of lung macrophage toward a pro-metastatic phenotype. Here, we show that multi-dose chemo treatment promotes breast cancer lung metastasis in the spontaneous breast cancer animal model. Pretreatment with Gemcitabine and Paclitaxel induces a significantly higher number of pulmonary metastases in the experimental metastasis model. Mechanistically, multi-dose of chemotherapy induces reactive myelopoiesis characterized by increase of BM LSK cells and multipotent progenitors in tumor-free mice, which is associated with the increase of mitochondrial ROS. Further, chemokine CCL2 expression is significantly increased in the plasma and lung tissues of chemo-treated tumor-free mice. Consequently, the percentage of BM Ly6C+ monocytes are significantly increased because of increased progenitor monocyte differentiation potential and monocyte proliferation, which results in an increase of lung monocytes and IMs with enhanced expression of coagulation factor X (FX). Inhibition of activated FX (FXa) can inhibit chemotherapy-induced lung metastasis. Together, these studies suggest a novel mechanism for chemotherapy induced metastasis via the modulation of BM-derived monocytes/macrophages and extravascular clotting in lung tissues. Supported by grants from NIH (P20GM135004)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel viral pathogen that causes a clinical disease called coronavirus disease 2019 (COVID-19). Approximately 20% of infected patients experience a severe manifestation of the disease, causing bilateral pneumonia and acute respiratory distress syndrome. Severe COVID-19 patients also have a pronounced coagulopathy with approximately 30% of patients experiencing thromboembolic complications. However, the etiology driving the coagulopathy remains unknown. Here, we explore whether the prominent neutrophilia seen in severe COVID-19 patients contributes to inflammation-associated coagulation. We found in severe patients the emergence of a CD16IntCD44lowCD11bInt low-density inflammatory band (LDIB) neutrophil population that trends over time with changes in disease status. These cells demonstrated spontaneous neutrophil extracellular trap (NET) formation, phagocytic capacity, enhanced cytokine production, and associated clinically with D-dimer and systemic IL-6 and TNF-α levels, particularly for CD40+ LDIBs. We conclude that the LDIB subset contributes to COVID-19-associated coagulopathy (CAC) and could be used as an adjunct clinical marker to monitor disease status and progression. Identifying patients who are trending towards LDIB crisis and implementing early, appropriate treatment could improve all-cause mortality rates for severe COVID-19 patients. One Sentence Summary In this study, we discover that low-density neutrophils significantly contribute to COVID-19-associated coagulopathy and inflammation

One of the defining characteristics of a pre-metastatic niche, a fundamental requirement for primary tumor metastasis, is infiltration of immunosuppressive macrophages. How these macrophages acquire their phenotype remains largely unexplored. Here, we demonstrate that tumor-derived exosomes (TDEs) polarize macrophages toward an immunosuppressive phenotype characterized by increased PD-L1 expression through NF-kB-dependent, glycolytic-dominant metabolic reprogramming. TDE signaling through TLR2 and NF-κB leads to increased glucose uptake. TDEs also stimulate elevated NOS2, which inhibits mitochondrial oxidative phosphorylation resulting in increased conversion of pyruvate to lactate. Lactate feeds back on NF-κB, further increasing PD-L1. Analysis of metastasis-negative lymph nodes of non-small-cell lung cancer patients revealed that macrophage PD-L1 positively correlates with levels of GLUT-1 and vesicle release gene YKT6 from primary tumors. Collectively, our study provides a novel mechanism by which macrophages within a pre-metastatic niche acquire their immunosuppressive phenotype and identifies an important link among exosomes, metabolism, and metastasis.

Ethnopharmacological relevance Epimedium koreanum Nakai is documented as tonic herbal in China for over a thousand years and has the potential to enhance the body's immunity according to the theory of traditional Chinese medicine. Polysaccharides are one of the most important effective compounds in Epimedium koreanum Nakai. Accumulating evidence indicated polysaccharides derived from traditional Chinese medicine have potent immune-enhancing properties and relatively nontoxic effects in cancer treatment. However, information about immunological regulation in tumor of Epimedium koreanum Nakai polysaccharides is limited and the reports of purification, characterization of polysaccharides have remained less. The purpose of our study was to further investigate the active polysaccharides from Epimedium koreanum Nakai by evaluating the immune-regulation activities in tumor-bearing mice and provide reasonable explanation for traditional application. Materials and methods We firstly purified Epimedium koreanum polysaccharide (EPS) from crude extracts and evaluated EPS in vitro using immunological experiments including maturation and Ag presentation function of DCs, CD4 T-cell differentiation and secretion of anti-cancer cytokines. In LLC-bearing mice model, we investigated its antitumor activities through evaluation of tumor cell proliferative activity, calculation of immune organ indexes and relative host immune system function tests. Results Results showed that EPS (180 × 104 Da) was composed of mannose (Man), rhamnose (Rha), glucuronic acid (GlcUA), galactosamine (GalN), glucose (Glc), galactose (Gal), arabinose (Ara) and fructose (Fuc). Chemical composition assay indicated EPS was a fraction with 28.20% uronic acid content. FT-IR suggested the presence of pyraoid ring in EPS and SEM displayed smooth surface embedded by several pores. Moreover, Our study suggested EPS could remarkably stimulate macrophages to secrete substantial anti-cancer cytokines and promote maturation as well as Ag presentation function of DCs. Strikingly, CD4 T-cell differentiation and increased INF-γ production stimulated by EPS–activated macrophages were observed in the research. Furthermore, EPS exhibited prominent antitumor activities through regulating host immune system function in LLC-bearing mice. Taken together, experimental findings suggested EPS could be regarded as a potential immune-stimulating modifier for cancer therapy. Conclusion Our studies demonstrated the polysaccharide (180 × 104 Da) purified from Epimedium koreanum Nakai could promote maturation and Ag presentation function of DCs, increase the level of immunomodulatory cytokines and activate CD4 T-cell differentiation. Furthermore, it may inhibit the tumor growth in LLC-bearing mice through regulating host immune system function

Regulations of myeloid-derived suppressor cells (MDSC) by ongoing inflammation following repeated chemotherapy remain elusive. Here, we show that multi-dose clinical regimen of Gemcitabine (GEM) treatment enhances the immunosuppressive function of monocytic (M)-MDSC although tumor development is delayed in E0771 tumor-bearing mice. Accordingly, effector IFN-γ-producing CD4 and CD8 T cells significantly decrease in the tumor microenvironment (TME) of GEM-treated mice. The conditioned medium (CM) of GEM-treated tumor cells enhances differentiation of mouse BM cells and human PBMC into immunosuppressive M-MDSC. Cytokine profiling of GEM-treated tumor cells identifies GM-CSF as one of the most differentially expressed cytokines. Blockade or knockdown of GM-CSF can partially reduce immunosuppression of Ly6C(high) cells induced by GEM-CM. Knockdown of GM-CSF in tumor cells also delays tumor progression with decreased accumulation of M-MDSC in TME. Mechanistically, enhanced production of reactive oxygen species (ROS) and activation of NF-κB are observed in GEM-treated tumor cells. Treatment with the mitochondrial targeted antioxidant and inhibitor of NF-κB signaling can abrogate GEM-induced hyperexpression of GM-CSF in E0771 cells. In addition, the phagocytic clearance of apoptotic tumor cells (efferocytosis) enhances the immunosuppressive function of BM Ly6C(high) myeloid cells. Further, GEM treatment results in metabolic changes in residual tumor cells leading to the resistance to T-cell mediated killing. Together, our results define an undesired effect of repeated GEM treatment promoting immunosuppression in TME via upregulation of GM-CSF and efferocytosis as well as deregulation of lipid metabolism in residual tumor cells.

The role of the mTOR signaling pathway in different myeloid cell subsets is poorly understood in the context of tumor development. In this study, myeloid cell–specific Raptor knockout (KO) mice were used to determine the roles of mechanistic target of rapamycin complex 1 (mTORC1) in regulating macrophage function from Lewis lung carcinoma (LLC) s.c. tumors and lung tumor metastasis. We found no difference in tumor growth between conditional Raptor KO and control mice in the s.c. tumor models, although depletion of mTORC1 decreased the immunosuppressive function of tumor-associated macrophages (TAM). Despite the decreased immunosuppressive activity of TAM, M1-like TAM differentiation was impaired in the s.c. tumor microenvironment of mTORC1 conditional Raptor KO mice due to downregulated CD115 expression on macrophages. In addition, TNF-α production by mTORC1-deficient myeloid cells was also decreased in the s.c. LLC tumors. On the contrary, disruption of mTORC1 in myeloid cells promoted lung cancer metastasis. Accordingly, immunosuppressive interstitial macrophages/metastasis-associated macrophages (CD11b+F4/80high) were accumulated in the lungs of Raptor KO mice in the LLC lung metastasis model, leading to decreased Th1 responses. Taken together, our results demonstrate that differential tumor microenvironment dictates the immunological outcomes of myeloid cells, with mTORC1 disruption leading to different tumor growth phenotypes.

Metastasis-associated macrophages (MAMs) play important roles in tumor metastasis through the formation of a pre-metastatic niche. In mouse models of lung metastasis, interstitial macrophages (IMs) markedly accumulate in the lungs and differentiate into MAMs. Regarding the origin of IMs/MAMs, tissue-resident macrophages and bone marrow (BM)-derived classical monocytes (BM-Mo) contribute to the pool of MAMs. Chemotherapy offers long-term clinical benefits to many cancer patients. However, several pre-clinical studies have demonstrated that certain cytotoxic drugs enhance metastasis by multiple mechanisms. Our recent studies suggest that Gemcitabine (GEM) promotes the immunosuppressive function of monocytic MDSC (M-MDSC) in the tumor microenvironment via the tumor-derived GM-CSF and efferocytosis signaling. In this study, we hypothesize that host responses to GEM treatment enhance the accumulation of BM-derived monocytes in lung, which promotes lung metastasis. Multi-dose of GEM treatment induces the increase of BM LSK cells and multipotent progenitors (MPPs) in tumor-free mice, which is associated with the increase of treatment induce mitochondrial ROS (mtROS). Thus, the percentage of BM Ly6C+ monocytes is significantly increased because of increased progenitor monocyte differentiation potential and monocyte proliferation. Further, GEM treatment increases the percentages of lung CCR2+Ly6C+ monocytes and IMs. Consequently, GEM pre-treated mice have a significantly higher number of pulmonary metastases compared to untreated mice in the experimental metastasis model. These results suggest a novel mechanism for chemotherapy induced metastasis via the modulation of myelopoiesis progenitors.

Antibody maturation as well as memory B and plasma cell differentiation occur primarily in the germinal centers (GC). Systemic lupus erythemotosus (SLE) may develop as a result of enhanced GC activity. Previous studies have shown that the dysregulated signal transducer and activator of transcription 3 (STAT3) pathway is linked to lupus pathogenesis. However, the exact role of STAT3 in regulating SLE disease progression has not been fully understood. Here, we demonstrated that the STAT3 signaling in B cells is essential for the GC formation and maintenance as well as antibody response. Increased cell apoptosis and down-regulated Bcl-xL and Mcl-1 anti-apoptotic gene expression were found in the STAT3 deficient GC B cells. The Tfh cell response positively correlated with the GC B cells and was significantly decreased in immunized B cell STAT3-deficient mice. STAT3 deficiency also led to the defect of plasma cell differentiation. Furthermore, STAT3 deficiency in autoreactive B cells resulted in decreased autoantibody production. Results obtained from B-cell STAT3 deficient B6.MRL/lpr mice suggest that STAT3 signaling significantly contributes to the SLE pathogenesis by regulation of the GC reactivity, autoantibody production, and kidney pathology. Our findings provide new insights into the role of STAT3 signaling in the maintenance of the GC formation and GC B cell differentiation and identify STAT3 as a novel target for the treatment of SLE.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that promote tumor progression. In this study, we demonstrated that activation of a C-type lectin receptor, dectin-1, in MDSC differentially modulates the function of different MDSC subsets. Yeast-derived whole β-glucan particles (WGP; a ligand to engage and activate dectin-1, oral treatment in vivo) significantly decreased tumor weight and splenomegaly in tumor-bearing mice with reduced accumulation of polymorphonuclear MDSC but not monocytic MDSC (M-MDSC), and decreased polymorphonuclear MDSC suppression in vitro through the induction of respiratory burst and apoptosis. On a different axis, WGP-treated M-MDSC differentiated into F4/80+CD11c+ cells in vitro that served as potent APC to induce Ag-specific CD4+ and CD8+ T cell responses in a dectin-1–dependent manner. Additionally, Erk1/2 phosphorylation was required for the acquisition of APC properties in M-MDSC. Moreover, WGP-treated M-MDSC differentiated into CD11c+ cells in vivo with high MHC class II expression and induced decreased tumor burden when inoculated s.c. with Lewis lung carcinoma cells. This effect was dependent on the dectin-1 receptor. Strikingly, patients with non–small cell lung carcinoma that had received WGP treatment for 10–14 d prior to any other treatment had a decreased frequency of CD14−HLA-DR−CD11b+CD33+ MDSC in the peripheral blood. Overall, these data indicate that WGP may be a potent immune modulator of MDSC suppressive function and differentiation in cancer.

In this study, an innovative type of hybrid beam–column joint using energy dissipated connection plates and I–shaped steel connectors to connect beams and columns was developed. Five interior beam–column specimens including a monolithic specimen and four hybrid precast joints, were tested under reversed cyclic loading to evaluate the seismic behavior of the connections. Test variables were the hybrid connection forms, flange cover plate, and steel fiber concrete usage. During the test, the hysteresis curves and cracking patterns were recorded. The seismic indicators, such as stiffness degradation, strength, energy dissipation, and ductility were determined. The test results revealed that the hybrid joint with the energy dissipated connection plates and steel fiber concrete failed in a typical flexural mode, exhibited stable hysteretic responses, and showed satisfactory seismic performance. The strength and cumulative energy dissipation of the proposed joint were higher by 46.6% and 230.9%, respectively, when compared with the monolithic specimen. The proposed hybrid connection could effectively transfer the plastic hinges to the connection region, reduce the damage and mitigate the stiffness deterioration due to the energy dissipated connection plates and the tensile deformability of the steel fiber. Therefore, the hybrid beam–column joints had superior performance to resist seismic loading. The developed seismic beam–column joints give a viable alternative for the precast concrete moment–resisting frames due to easy construction.

Despite the remarkable success of immunotherapy, pancreatic cancer has yet to benefit. This creates a desperate need for therapies that target pancreatic cancer and utilize novel approaches to prevent and treat this lethal disease. Innate immune cells are vital in antitumor immunosurveillance. Recent studies have shown that innate immune cells possess a form of memory termed Trained Immunity (TI), where cells exposed to an initial stimulus undergo epigenetic and metabolic reprograming that causes hyper-responsiveness when exposed to a second signal. It is yet unknown whether inducing TI could protect against tumors, and specifically pancreatic tumors. Here we show that yeast-derived particulate β-glucan, a known TI inducer, results in 90% of an intraperitoneal dose trafficking to the pancreas. This leads to a robust, dose-dependent influx of innate and adaptive immune cells into the pancreas that have enhanced Th1 function. Despite the cellular influx, signs of pancreatitis are absent. In vivo and in vitro studies of β-glucan trained pancreatic macrophages show increased TNFα and IL-6 production when exposed to LPS, pancreatic tumor cells and their supernatant. This indicates that pancreatic macrophages can be trained to protect against pancreatic tumor cells. Depletion of various immune populations suggest that pancreatic macrophages are directly trained by β-glucan, which then stimulate NK and T-cells. In an orthotopic model of pancreatic cancer, β-glucan trained mice show reduced tumor burden and augmented antitumor functions of innate and adaptive cells. Together, this suggests that β-glucan can train pancreatic innate immune cells to have antitumor memory, which can serve as a novel approach to treating pancreatic cancer.

A new type of precast hybrid steel fiber concrete (SFC)/reinforced concrete (RC) beam–column connection with different connection details was developed for precast concrete frames. In this study, seven concrete beam–column connections, six precast specimens, and one monolithic joint were subjected to a constant axial load and a reversed cyclic loading. The main test variables were the end-plate connection forms and SFC usage in the core region and the connection section. The construction process of the proposed hybrid connections was convenient and fast. The load–displacement hysteresis curves and failure modes of the precast connections were obtained, and the seismic behaviors in terms of connection stiffness, strength, energy dissipation, and shear deformation in the joint core were evaluated. The results revealed that the proposed precast concrete connections had higher strength, slower stiffness degradation, and greater energy dissipation capacity, thereby illustrating that the specimens had a satisfactory ability to resist seismic loads. The shear deformation of the proposed joints was reduced because of the tensile deformability of the SFC and the yielding steel connectors, while the shear strength was considerably enhanced. The cracking pattern in the core region of the hybrid connections with the SFC was well controlled. The integrity of the connection section was maintained. Furthermore, the SFC and steel-plate connectors were vital to the performance of the system.

A mutation in STAT3 has been linked to the incidence of autosomal dominant hyper IgE syndrome, a disease characterized by elevated serum IgE Ab. However, how this genetic mutation leads to the phenotype has not been fully understood. We investigated the specific role of STAT3 in the germinal center (GC) B cells and plasma cells for IgE class switching. Through the use of STAT3 conditional knockout (cKO) mice in a Th2-type immunization model, we demonstrated that CD2-Cre–driven STAT3 cKO mice showed elevated IgE and decreased IgG1 in the serum and a reduction in GC formation. Within the GC, IgG1+ GC B cells were decreased, whereas IgE+ GC B cells were more prevalent. Additionally, these mice exhibited reduced IgG1 and elevated IgE populations of Ab-producing plasma cells. Subsequent experiments using a CD19-Cre cKO mouse established this effect to be B cell–intrinsic. Transcription factors critical for GC and plasma cell differentiation, including Bcl-6 and Aicda, were shown to function as downstream signals of STAT3 regulation. Chromatin immunoprecipitation sequencing analysis revealed that many genes, including Bcl3 and Crtc2, were among the direct STAT3 regulated targets. Mice with STAT3 deficiency in B cells also demonstrated an increase in lung inflammation when used in an asthma-like disease model. This model suggests a negative role for STAT3 in regulating class switching of the GC B cells from the IgG1 to the IgE producing state, which may serve as a therapeutic target for treatment of autosomal dominant hyper IgE syndrome and other immune disorders.

Some chemotherapeutic agents can selectively deplete MDSC in vivo. However, recent studies suggest that Gemcitabine (GEM) and 5-Fluorouracil (5-FU) treatment decrease the anticancer efficacy by activating the inflammasome pathway in MDSC. In addition, chemotherapy can modify the tumor microenvironment by induction of chronic inflammation. Therefore, regulations of MDSC by chemotherapy still remain elusive. Here, we explored the mechanisms by which multi-dose clinical regimen of GEM treatment induce an ongoing inflammation leading to the enhanced immunosuppressive activity of monocytic MDSC (M-MDSC). We found that repeated GEM treatment promoted the expansion and differentiation of immunosuppressive M-MDSC although tumor development was delayed in E0771 tumor-bearing mice. Accordingly, IFN-γ-producing CD4 and CD8 T cells were significantly decreased in the TME of GEM-treated mice. The conditioned medium of GEM-treated tumor cells (GEM-CM) promoted the differentiation of immunosuppressive mouse BM Ly6Chigh cells. Tumor-derived soluble factors, such as GM-CSF and soluble ICAM-1(sICAM-1), were up-regulated upon chemo-drug treatment. Blocking of GM-CSF or ICAM-1 with neutralizing Abs could partially reduce immunosuppression of Ly6Chigh cells induced by GEM-CM. Enhanced production of reactive oxygen species (ROS) and activation of NF-κB were observed in GEM-treated tumor cells. Treatment with the inhibitor of NF-κB signaling could abrogate GEM-induced hyperexpression of GM-CSF in E0771 cells. Together, our results define an undesired effect of GEM-induced ROS which leads to NF-κB activation, up-regulation of GM-CSF, and subsequent differentiation of immunosuppressive myeloid cells.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that promote tumor progression. Herein, we demonstrated that activation of a C-type lectin receptor, dectin-1, in MDSC differentially modulates the function of different MDSC subsets. Yeast-derived whole β-glucan particles (WGP), a ligand to engage and activate dectin-1, oral treatment in vivo significantly decreased tumor weight and splenomegaly in tumor-bearing mice with reduced accumulation of PMN-MDSC but not M-MDSC, and decreased PMN-MDSC suppression in vitro through the induction of respiratory burst and apoptosis. On a different axis, WGP-treated M-MDSC differentiated into F4/80+CD11c+ cells in vitro that served as potent antigen-presenting cells (APC) to induce Ag-specific CD4+ and CD8+ T cell responses in a dectin-1 dependent manner. In addition, ERK1/2 phosphorylation was required for the acquisition of APC properties in M-MDSC. Moreover, WGP-treated M-MDSC differentiated into CD11c+ cells in vivo with high MHC class II expression and induced decreased tumor burden when inoculated subcutaneously with LLC cells. This effect was dependent of the dectin-1 receptor. Strikingly, patients with non-small cell lung cancer (NSCLC) that had received WGP treatment for 10–14 days prior to any other treatment had a decreased frequency of CD14−HLA-DR−CD11b+CD33+ MDSC in the peripheral blood. Overall, these data indicate that WGP may be a potent immune modulator of MDSC suppressive function and differentiation in cancer.

Ab therapy against surface Ags on tumor cells has demonstrated significant efficacy for some cancers. However, it is costly and patients frequently develop acquired resistance over time. In cases of Ab therapy resistance, T cell responses have been shown to be essential in controlling disease progression. Thus, vaccination that generates a sustained Ab response as well as a T cell response may be more effective and economical. Here, we have developed a vaccination strategy by targeting protein Ags to B cells via a CD19 single chain variable fragment miniAb. By using the tumor-associated Ag (TAA) her-2/neu extracellular domain (ECD), we showed that the co-engagement of CD19 and BCR induced full B cell activation to produce a high titer of Abs and enhanced CD4 Th2 response and CD8 T cell activation and differentiation. These Abs competitively inhibited humanized her-2/neu Ab binding and were capable of activating the complement and inhibiting human breast cancer growth in vitro. Therapeutic efficacy was demonstrated in vivo using murine mammary carcinomas models. Furthermore, four different ECDs of her-2/neu could be targeted to B cells to generate Abs against particular domains with different anti-tumor properties. This approach may offer a new avenue for vaccine development with significantly lower cost which may be usable not only for cancer therapy but also for infectious agents.

Tumor-associated macrophages (TAM) with an M2-like phenotype have been linked to tumor-elicited inflammation, immunosuppression, and resistance to chemotherapies in cancer, thus representing an attractive target for an effective cancer immunotherapy. Here, we demonstrate that particulate yeast-derived β-glucan, a natural polysaccharide compound, converts polarized M2 macrophages or immunosuppressive TAM into an M1-like phenotype with potent immuno-stimulating activity. This process is associated with macrophage metabolic reprograming with enhanced glycolysis, krebs cycle and glutamine utilization. In addition, particulate β-glucan converts immunosuppressive TAM via the C-type lectin receptor dectin-1-induced Syk-Card9-Erk pathway. Further in vivo studies show that oral particulate β-glucan treatment significantly delays tumor growth, which is associated with in vivo TAM phenotype conversion and enhanced effector T cell activation. Mice injected with particulate β-glucan-treated TAM mixed with tumor cells have significantly reduced tumor burden with less blood vascular vessels compared to those with TAM plus tumor cell injection. In addition, macrophage depletion significantly reduced the therapeutic efficacy of particulate β-glucan in tumor-bearing mice. These findings have established a new paradigm for macrophage polarization and immunosuppressive TAM conversion and shed the light on the action mode of β-glucan treatment in cancer.

Nature Communications 5: Article number: 3986 (2014); Published 9 June 2014; Updated 11 April 2016 The financial support for this Article was not fully acknowledged. The Acknowledgements should have included the following: Huang-Ge Zhang is supported by a Research Career Scientist (RCS) Award.

Purpose: The beneficial properties of β-glucans have been recognized for centuries. Their proposed mechanisms of action in cancer therapy occur via stimulation of macrophages and priming of innate neutrophil complement receptor 3 for eliciting complement receptor 3–dependent cellular cytotoxicity of iC3b-opsonized tumor cells. The current study is to investigate whether β-glucan therapy has any effect on antitumor adaptive T-cell responses. Experimental Design: We first examined the trafficking of orally administered particulate yeast-derived β-glucan and its interaction with dendritic cells (DC) that captured tumor materials. Antigen-specific T cells were adoptively transferred into recipient mice to determine whether oral β-glucan therapy induces augmented T-cell responses. Lewis lung carcinoma and RAM-S lymphoma models were used to test oral β-glucan therapeutic effect. Further mechanistic studies including tumor-infiltrating T cells and cytokine profiles within the tumor milieu were determined. Results: Orally administered particulate β-glucan trafficked into spleen and lymph nodes and activated DCs that captured dying tumor cells in vivo, leading to the expansion and activation of antigen-specific CD4 and CD8 T cells. In addition, IFN-γ production of tumor-infiltrating T cells and CTL responses were significantly enhanced on β-glucan treatment, which ultimately resulted in significantly reduced tumor burden. Moreover, β-glucan–treated tumors had significantly more DC infiltration with the activated phenotype and significant levels of Th1-biased cytokines within the tumor microenvironment. Conclusions: These data highlight the ability of yeast-derived β-glucan to bridge innate and adaptive antitumor immunity and suggest that it can be used as an adjuvant for tumor immunotherapy. Clin Cancer Res; 16(21); 5153–64. ©2010 AACR.

Sensitization to major histocompatibility complex (MHC) alloantigens is critical in transplantation rejection. The mechanism of sensitization to minor histocompatibility antigens (Mi-HAg) has not been thoroughly explored. We used a mouse model of allosensitization to Mi-HAg to study the Mi-HAg sensitization barrier in bone marrow transplantation (BMT). AKR mice were sensitized with MHC congenic Mi-HAg disparate B10.BR skin grafts. Adaptive humoral (B-cells) and cellular (T cells) responses to Mi-HAg are elicited. In subsequent BMT, only 20% of sensitized mice engrafted, while 100% of unsensitized mice did. In vivo cytotoxicity assays showed that Mi-HAg sensitized AKR mice eliminated CFSE labeled donor splenocytes significantly more rapidly than naïve AKR mice but less rapidly than MHC-sensitized recipients. Sera from Mi-HAg sensitized mice also reacted with cells from other mouse strains, suggesting that Mi-HAg peptides were broadly shared between mouse strains. The production of anti-donor-Mi-HAg antibodies was totally prevented in mice treated with anti-CD154 during skin grafting, suggesting a critical role for the CD154:CD40 pathway in B-cell reactivity to Mi-HAg. Moreover, anti-CD154 treatment promoted BM engraftment to 100% in recipients previously sensitized to donor Mi-HAg. Taken together, Mi-HAg sensitization poses a significant barrier in BMT and can be overcome with CD154:CD40 costimulatory blockade.

A mutation in the Signal Transducer and Activator of Transcription 3 (STAT-3) has been linked to incidence of Autosomal Dominant Hyper Immunoglobulin E Syndrome (AD-HIES), a disease characterized by elevated serum IgE antibody and susceptibility to respiratory and skin infection. However, how this genetic mutation leads to the phenotype has not been fully understood. In this study we investigated the specific role of STAT-3 in the germinal center (GC) B cells and plasma cells for IgE class switching. Through the use of CD2-Cre or CD19-Cre driven STAT-3 conditional knockout (cKO) mice in a Th2-type immunization model, we aimed to determine the specific cell subset(s) and signaling mechanism by which STAT-3 mediated class switching is regulated. We demonstrated in vivo and in vitro that CD2-Cre driven STAT-3 cKO mice showed elevated IgE and decreased IgG1 in the serum, and a reduction in GC formation. Within the GC, IgG1+ GC B cells were decreased while IgE+ GC B cells were more prevalent. Additionally, these mice exhibited reduced IgG1 and elevated IgE populations of antibody-producing plasma cells. Subsequent experiments using a CD19-Cre B-cell specific cKO mouse established this effect to be B-cell intrinsic. Mice with STAT-3 deficiency in B cells have also demonstrated an increase in lung inflammation when used in an asthma-like disease model. Our data also revealed other transcription factors, including Bcl-6 and Aicda, function as downstream signals of STAT-3 regulation. This model suggests a negative role for STAT-3 in regulating class switching of the GC B cells from the IgG1 to the IgE producing state, which may serve as a therapeutic target for treatment of AD-HIES and other immune disorders.

The mTOR inhibitors are used in clinics for treating multiple cancers. However, this assertion does not fully address the effects on the tumor microenvironment (TME) which plays critical roles in cancer development and metastasis. Moreover, a controversy exists in the literature regarding the roles of mTOR in regulating myeloid cell activation in response to different environmental factors. In this study, conditional Raptor KO mice were generated to disrupt mTORC1 signaling in myeloid cells and LLC tumor cells were implanted either s.c. or i.v. in syngeneic mice. Although depletion of mTORC1 signaling promoted M2 macrophages and TAM toward an M1-like phenotype with decreased immunosuppressive activity, tumor progression in the s.c. model didn’t show difference between Raptor KO and control mice. Flow cytometric analysis demonstrated decreased Th1 T cell response in the TME of Raptor KO mice. The depletion of mTORC1 signaling decreased TAM trafficking and increased Ly6C+accumulation in LLC TME, which was correlated with lower levels of CD115 expression on Raptor-deficient macrophages. Cytokine profile analysis revealed that TNF-a secretion by myeloid cells was significantly decreased in Raptor-deficient TME. Further, disruption of mTORC1 in myeloid cells promoted lung cancer metastasis, which was associated with decreased Th1 and NK cell response. Accordingly, more CD11b+F4/80high macrophages were accumulated in the lungs of Raptor KO mice in LLC metastasis model. Taken together, our results demonstrate that mTORC1signaling in myeloid cells may have multiple roles in regulating anti-tumor immunity dependent on tumor environment, specifically with pro- metastatic effects in mTORC1-deficient lung myeloid cells.

Plasmacytoid dendritic cells (pDCs) are specialized type I IFN producers, which play an important role in pathogenesis of autoimmune disorders. Dysregulated autoreactive B cell activation is a hallmark in most autoimmune diseases. This study was undertaken to investigate interactions between pDCs and autoreactive B cells. After coculture of autoreactive B cells that recognize self-Ag small nuclear ribonucleoprotein particles with activated pDCs, we found that pDCs significantly enhance autoreactive B cell proliferation, autoantibody production, and survival in response to TLR and BCR stimulation. Neutralization of IFN-α/β and IL-6 abrogated partially pDC-mediated enhancement of autoreactive B cell activation. Transwell studies demonstrated that pDCs could provide activation signals to autoreactive B cells via a cell-to-cell contact manner. The involvement of the ICAM-1-LFA-1 pathway was revealed as contributing to this effect. This in vitro enhancement effect was further demonstrated by an in vivo B cell adoptive transfer experiment, which showed that autoreactive B cell proliferation and activation were significantly decreased in MyD88-deficient mice compared with wild-type mice. These data suggest the dynamic interplay between pDCs and B cells is required for full activation of autoreactive B cells upon TLR or BCR stimulation.

B cells are antibody (Ab)–secreting cells as well as potent antigen (Ag)–presenting cells that prime T-cell activation, which evokes great interest in their use for vaccine development. Here, we targeted ovalbumin (OVA) to B cells via CD19 and found that a single low dose of anti–CD19-OVA conjugates, but not isotype mAb-OVA, stimulated augmented CD4 and CD8 T-cell proliferation and expansion. Administration of TLR9 agonist CpG could significantly enhance long-term T-cell survival. Similar results were obtained when the tumor-associated Ag MUC1 was delivered to B cells. MUC1 transgenic (Tg) mice were previously found to lack effective T-cell help and produce low-titer of anti-MUC1 Abs after vaccination. Targeting MUC1 to B cells elicited high titer of anti-MUC1 Abs with different isotypes, predominantly IgG2a and IgG2b, in MUC1 Tg mice. The isotype switching of anti-MUC1 Ab was CD4 dependent. In addition, IFN-γ–producing CD8 T cells and in vivo cytolytic activity were significantly increased in these mice. The mice also showed significant resistance to MUC1+ lymphoma cell challenge both in the prophylactic and therapeutic settings. We conclude that Ags targeting to B cells stimulate CD4 and CD8 T-cell responses as well as Th-dependent humoral immune responses.

The therapeutic benefits of fungal β-glucans have been demonstrated as immuno-stimulating agents. In this study, we aimed to explore the mechanisms used by yeast β-glucan-rich particles to activate murine resident macrophages for cytokine secretion. We demonstrated that resident macrophages were effectively activated by whole yeast β-glucan particles (WGPs), such as with the upregulation of co-stimulatory molecules and the secretion of cytokines. The binding ability of WGPs and the levels of cytokine secretion in resident macrophages were significantly inhibited by soluble yeast β-glucan but not by blockade of zymosan glucan receptor dectin-1. In addition, WGP-stimulated cytokine secretion was partially dependent on the MyD-88 pathway but was not significantly affected in CR3-deficient (CR3−/−) mice. Furthermore, we showed that Syk kinase was recruited upon WGP stimulation and was required for the production of cytokines. Taken together, these observations suggest that β-glucan recognition is necessary but not sufficient to induce inflammatory response on resident macrophages. In addition, β-glucan particles may use differential mechanisms for cytokine secretion in resident macrophages that may modulate both innate and adaptive immunity.

It has become clear that the autoreactive B cells are a part of the normal naïve B cell repertoire in the periphery, despite the fact that they undergo a series of checkpoints, which include receptor editing (revision), clonal deletion, and anergy. However, most of those B cells reactive against self antigen remain functionally naïve for autoantibody production by differential peripheral checkpoints. Therefore, the presence of autoreactive B cells does not always signify disease. Regulation of their activation and effector functions will determine the ultimate outcome. Although autoreactive B cell tolerance is well maintained in the healthy individual, the existence of pathogenic autoantibodies in autoimmune diseases indicates that these tolerogenic checkpoints are broken. Recent studies have demonstrated that autoreactive B cells are regulated by a composite of factors, such as genetic susceptibility and environmental triggers such as bacterial and viral infections as well as other immune cells. Interestingly, Toll-like receptors, previously considered as pattern-recognition receptors to detect and sense pathogens, may also have a potential to recognize self antigens and regulate autoreactive B cells for activation. Understanding the mechanisms of autoreactive B cell regulation and activation may help in identifying novel targets for the treatment of autoimmune diseases.

Autoreactive B cells are the source of pathogenic autoantibodies (autoAb) in systemic lupus erythematosus (SLE). Previous studies have demonstrated that anti-small nuclear ribonucleoprotein particles (snRNP) B cells from normal background mice tolerize T cells in the periphery and do not secrete autoAb. In this study, we examined whether these anti-snRNP B cells can be activated for autoAb production by the engagement of Toll-like receptors (TLR). Anti-snRNP B cells proliferated vigorously and secreted abundant anti-snRNP autoAb upon exposure to CpG or polyriboinosinic polyribocytidylic acid [poly (I:C)] in vitro. In addition, the costimulatory molecules CD80 and CD86 were up-regulated. While both anti-snRNP B cells and wild-type B cells produced similar levels of IL-6 and IL-10, anti-snRNP B cells secreted predominately IFN-gamma in response to CpG or poly (I:C) stimulation. Furthermore, we showed that in vivo engagement of TLR stimulated immature anti-snRNP B cells to further differentiate and produce autoAb and form germinal centers. The activated anti-snRNP B cells became expanded and migrated into the T-B cell interface. Moreover, TLR engagement directly or indirectly activated autoreactive B cells via a CD4 T cell-independent manner. These results provide in vitro and in vivo evidence that BCR/TLR co-engagement promotes the activation of anti-snRNP B cells for autoAb production.

Anti-tumor mAbs hold promise for cancer therapy, but are relatively inefficient. Therefore, there is a need for agents that might amplify the effectiveness of these mAbs. One such agent is β-glucan, a polysaccharide produced by fungi, yeast, and grains, but not mammalian cells. β-Glucans are bound by C receptor 3 (CR3) and, in concert with target-associated complement fragment iC3b, elicit phagocytosis and killing of yeast. β-Glucans may also promote killing of iC3b-opsonized tumor cells engendered by administration of anti-tumor mAbs. In this study, we report that tumor-bearing mice treated with a combination of β-glucan and an anti-tumor mAb show almost complete cessation of tumor growth. This activity evidently derives from a 25-kDa fragment of β-glucan released by macrophage processing of the parent polysaccharide. This fragment, but not parent β-glucan, binds to neutrophil CR3, induces CBRM 1/5 neoepitope expression, and elicits CR3-dependent cytotoxicity. These events require phosphorylation of the tyrosine kinase, Syk, and consequent PI3K activation because β-glucan-mediated CR3-dependent cytotoxicity is greatly decreased by inhibition of these signaling molecules. Thus, β-glucan enhances tumor killing through a cascade of events, including in vivo macrophage cleavage of the polysaccharide, dual CR3 ligation, and CR3-Syk-PI3K signaling. These results are important inasmuch as β-glucan, an agent without evident toxicity, may be used to amplify tumor cell killing and may open new opportunities in the immunotherapy of cancer.

Innate γδ T cells play critical roles in mucosal immunity such as regulating intestinal epithelial homeostasis. In addition, γδ T cells are significantly increased in the inflamed mucosa of patients with ulcerative colitis. However, γδ T cells are a heterogeneous population. IL-17-producing versus IFNγ-producing γδ T cells play differential roles in different disease settings. Therefore, dissecting the exact role of different subsets of γδ T cells in colitis is essential for understanding colitis immunopathogenesis. In the current study, we found that TCR δ-deficient mice had a more severe dextran sodium sulfate (DSS)-induced colitis that was reduced upon reconstitution of γδT17 cells but not IFNγ-producing γδ T cells. Immunophenotyping of the cellular infiltrate upon DSS-induced colitis showed a reduced infiltration of Gr-1+CD11b+ myeloid cells into the sites of inflammation in mice lacking γδT17 cells. Further experiments demonstrated that IL-17, IL-18, and chemokine CXCL5 were critical in Gr-1+CD11b+ myeloid cell recruitment. In vitro T cell suppressive assay indicated that this Gr-1+CD11b+ population was immunosuppressive. Depletion of Gr-1+CD11b+ myeloid cells resulted in an increase severity of DSS-induced colitis. Our study elucidates a new immune pathway involving γδT17-dependent recruitment of Gr-1+CD11b+ myeloid cells to the site of colitis inflammation important in the protection of colitis initiation and progression.

Dermal IL-17-producing γδT cells have a critical role in skin inflammation. However, their development and peripheral regulation have not been fully elucidated. Here we demonstrate that dermal γδT cells develop from the embryonic thymus and undergo homeostatic proliferation after birth with diversified TCR repertoire. Vγ6T cells are bona fide resident, but precursors of dermal Vγ4T cells may require extrathymic environment for imprinting skin-homing properties. Thymic Vγ6T cells are more competitive than Vγ4 for dermal γδT cell reconstitution and TCRδ(-/-) mice reconstituted with Vγ6 develop psoriasis-like inflammation after IMQ-application. Although both IL-23 and IL-1β promote Vγ4 and Vγ6 proliferation, Vγ4 are the main source of IL-17 production that requires IL-1 signalling. Mice with deficiency of IL-1RI signalling have significantly decreased skin inflammation. These studies reveal a differential developmental requirement and peripheral regulation for dermal Vγ6 and Vγ4 γδT cells, implying a new mechanism that may be involved in skin inflammation.