Exosome-like nanoparticles from Mulberry bark prevent DSS-induced colitis via the AhR/COPS8 pathway.

Bark protects the tree against environmental insults. Here, we analyzed whether this defensive strategy could be utilized to broadly enhance protection against colitis. As a proof of concept, we show that exosome-like nanoparticles (MBELNs) derived from edible mulberry bark confer protection against colitis in a mouse model by promoting heat shock protein family A (Hsp70) member 8 (HSPA8)-mediated activation of the AhR signaling pathway. Activation of this pathway in intestinal epithelial cells leads to the induction of COP9 Constitutive Photomorphogenic Homolog Subunit 8 (COPS8). Utilizing a gut epithelium-specific knockout of COPS8, we demonstrate that COPS8 acts downstream of the AhR pathway and is required for the protective effect of MBELNs by inducing an array of anti-microbial peptides. Our results indicate that MBELNs represent an undescribed mode of inter-kingdom communication in the mammalian intestine through an AhR-COPS8-mediated anti-inflammatory pathway. These data suggest that inflammatory pathways in a microbiota-enriched intestinal environment are regulated by COPS8 and that edible plant-derived ELNs may hold the potential as new agents for the prevention and treatment of gut-related inflammatory disease.

Anti-PD-1 antibody-activated Th17 cells subvert re-invigoration of antitumor cytotoxic T-lymphocytes via myeloid cell-derived COX-2/PGE2.

Anti-PD-1 antibody-mediated activation of type 17 T-cells undermines checkpoint inhibitor therapy in the LSL-KrasG12D murine lung cancer model. Herein, we establish that the Th17 subset is the primary driver of resistance to therapy demonstrate that the ontogeny of dysplasia-associated Th17 cells is driven by microbiota-conditioned macrophages; and identify the IL-17-COX-2-PGE2 axis as the mediator of CD8+ cytotoxic T-lymphocyte de-sensitization to checkpoint inhibitor therapy. Specifically, anti-PD-1 treatment of LSL-KrasG12D mice, in which CD4+ T-cells were deficient for RORc, resulted in a 60% increase in CTL cytotoxicity and a 2.5-fold reduction in tumor burden confirming the critical role of Th17 cells in resistance to therapy. Lung-specific depletion of microbiota reduced Th17 cell prevalence and tumor burden by 5- and 2.5-fold, respectively; establishing a link between microbiota and Th17 cell-driven tumorigenesis. Importantly, lung macrophages from microbiota sufficient, but not from microbiota-deficient, mice polarized naïve CD4+ T-cells to a Th17 phenotype, highlighting their role in bridging microbiota and Th17 immunity. Further, treatment with anti-PD-1 enhanced COX-2 and PGE2 levels, whereas neutralization of IL-17 diminished this effect. In contrast, inhibition of COX-2 rescued CTL activity and restored tumor suppression in anti-PD-1-treated mice, revealing the molecular basis of IL-17-mediated resistance to checkpoint blockade. Clinical implications of these findings are discussed.

Anti-PD-1 antibody-mediated activation of type 17 T-cells undermines checkpoint blockade therapy.

Tumors that develop in the genetic LSL-K-rasG12D murine lung cancer model are resistant to anti-PD-1 antibody treatment. Analysis of tumor-bearing lungs from anti-PD-1-treated mice revealed an up to 2.5-fold increase in IL-17-producing T-cells, with minimal change in CD8+ T-cell activity. Neutralization of IL-17 concurrent with anti-PD-1 treatment on the other hand, resulted in robust CD8+ T-cell activation and a threefold reduction in tumor burden. Loss-of-function studies demonstrated that anti-PD-1 driven activation of CD4+ and γδTCR+ T-cells contributed to IL-17-mediated de-sensitization of CD8+ cytotoxic T-cells (CTL) to therapy; and that CTL activation was critical to tumor eradication. Importantly, post-therapy lung Th17 cell prevalence and activity prognosticated treatment efficacy. Consistent with the murine data, analysis of tumor biopsy samples from non-small cell lung cancer (NSCLC) patients revealed that pre-therapy intratumoral CD8+/RORc+ cell ratio correlated with response to immune checkpoint blockade (ICB). These findings provide the initial evidence for a new mechanism of ICB resistance in lung cancer.

Temporospatial shifts within commercial laboratory mouse gut microbiota impact experimental reproducibility.

BACKGROUND: Experimental reproducibility in mouse models is impacted by both genetics and environment. The generation of reproducible data is critical for the biomedical enterprise and has become a major concern for the scientific community and funding agencies alike. Among the factors that impact reproducibility in experimental mouse models is the variable composition of the microbiota in mice supplied by different commercial vendors. Less attention has been paid to how the microbiota of mice supplied by a particular vendor might change over time. RESULTS: In the course of conducting a series of experiments in a mouse model of malaria, we observed a profound and lasting change in the severity of malaria in mice infected with Plasmodium yoelii; while for several years mice obtained from a specific production suite of a specific commercial vendor were able to clear the parasites effectively in a relatively short time, mice subsequently shipped from the same unit suffered much more severe disease. Gut microbiota analysis of frozen cecal samples identified a distinct and lasting shift in bacteria populations that coincided with the altered response of the later shipments of mice to infection with malaria parasites. Germ-free mice colonized with cecal microbiota from mice within the same production suite before and after this change followed by Plasmodium infection provided a direct demonstration that the change in gut microbiota profoundly impacted the severity of malaria. Moreover, spatial changes in gut microbiota composition were also shown to alter the acute bacterial burden following Salmonella infection, and tumor burden in a lung tumorigenesis model. CONCLUSION: These changes in gut bacteria may have impacted the experimental reproducibility of diverse research groups and highlight the need for both laboratory animal providers and researchers to collaborate in determining the methods and criteria needed to stabilize the gut microbiota of animal breeding colonies and research cohorts, and to develop a microbiota solution to increase experimental rigor and reproducibility.

Cytokine-Encapsulated Biodegradable Microspheres for Immune Therapy.

Therapeutic macromolecules such as proteins are conventionally administered via bolus injection, an approach that presents significant limitations. Sustained-release biodegradable nano/microsphere depots, on the other hand, represent a more physiological delivery tool for biologics. Here, we focus on an emerging novel application of this technology, i.e., cytokine-encapsulated biodegradable microspheres as immune therapeutics. The overall pre-clinical experience, recent advances and the clinical potential of such formulations are discussed.

Microspheres Encapsulating Immunotherapy Agents Target the Tumor-Draining Lymph Node in Pancreatic Ductal Adenocarcinoma.

INTRODUCTION: The tumor-draining lymph node (TDLN) plays a role in tumor immunity. Intratumorally administered microspheres (MS) that encapsulate immunomodulatory agents have emerged as a treatment strategy capable of causing profound changes in the tumor microenvironment (TME) and eliciting potent antitumor effects. We hypothesized that local delivery of MS to the TME may also drain to and therefore target the TDLN to initiate antitumor immune responses. METHODS: Fluorescent MS were injected into orthotopically implanted murine pancreatic tumors, and tissues were examined by whole-mount microscopy and imaging flow cytometry. The role of the TDLN was investigated for mice treated with intratumoral interleukin-12 (IL-12)-encapsulated MS in combination with stereotactic body radiotherapy (SBRT) by cytokine profile and TDLN ablation. RESULTS: Fluorescent AF-594 MS delivered intratumorally were detected in the tumor, peritumoral lymphatics, and the TDLN 2 h after injection. Phagocytic cells were observed with internalized fluorescent MS. SBRT + IL-12 MS-induced upregulation of Th1 and antitumor factors IL-12, IFN-γ, CXCL10, and granzyme B in the TDLN, and excision of the TDLN partially abrogated treatment efficacy. CONCLUSIONS: Our results demonstrate that intratumorally administered MS not only target the TME, but also drain to the TDLN. Furthermore, MS encapsulated with a potent antitumor cytokine, IL-12, induce an antitumor cytokine profile in the TDLN, which is essential for treatment efficacy.

Inhaled IL-10 Suppresses Lung Tumorigenesis via Abrogation of Inflammatory Macrophage-Th17 Cell Axis.

Intratracheal administration of a novel IL-10 formulation suppressed IL-17-driven, CD4+ T cell-dependent tumorigenesis in the LSL-K-rasG12D murine lung cancer model. Analysis of lung lymphocyte populations demonstrated that antitumor activity of IL-10 was associated with a 5-fold decline in Th17 cell prevalence and a concurrent suppression of inflammatory M1-like macrophage activity. Further phenotypic characterization revealed that macrophages and dendritic cells, but not Th17 cells, expressed IL-10RA on the cell surface with the CD11b+F4/80+CX3CR1+ interstitial macrophages representing the dominant IL-10RA+ subset. Consistent with these observations, in vitro stimulation of sorted CD4+ T cells with IL-10 did not affect their ability to produce IL-17, whereas similar treatment of purified interstitial macrophages resulted in a dramatic M1 to M2 phenotypic switch. Importantly, preconditioning of macrophages (but not of CD4+ T cells) with IL-10 led to potent suppression of CD4+ T cell IL-17 production in an in vitro coculture assay, suggesting that IL-10 suppressed Th17 cell activity primarily via its upstream effects on macrophages. In support of this notion, in vivo macrophage depletion resulted in a 5-fold decline in Th17 cell numbers and a concurrent 6-fold reduction in tumor burden. Collectively, these data demonstrate that in the LSL-K-rasG12D murine lung cancer model, inflammatory macrophage-Th17 cell axis is critical to tumorigenesis and that IL-10 blocks this process primarily via a direct effect on the former. Inhaled IL-10 formulations may be of use in prophylaxis against lung cancer in high-risk patients.

Tolerogenic Phenotype of IFN-γ-Induced IDO+ Dendritic Cells Is Maintained via an Autocrine IDO-Kynurenine/AhR-IDO Loop.

Previous studies demonstrated that IL-12-driven antitumor activity is short-circuited by a rapid switch in dendritic cell (DC) function from immunogenic to tolerogenic activity. This process was dependent on IFN-γ and the tolerogenic phenotype was conferred by IDO. Extended monitoring of IDO(+) DC in the tumor-draining lymph nodes of IL-12 plus GM-CSF-treated tumor-bearing mice revealed that whereas IFN-γ induction was transient, IDO expression in DC was maintained long-term. An in vitro system modeling the IFN-γ-mediated change in DC function was developed to dissect the molecular basis of persistent IDO expression in post-IL-12 DC. Stimulation of DC with IFN-γ and CD40L resulted in rapid induction of IDO1 and IDO2 transcription and recapitulated the in vivo switch from immunogenic to tolerogenic activity. Long-term maintenance of IDO expression was found to be independent of exogenous and autocrine IFN-γ, or the secondary cytokines TGF-ß, TNF-α, and IL-6. In contrast, both IDO enzymatic activity and IFN-γ-induced AhR expression were required for continued IDO transcription in vitro and in vivo. Addition of the tryptophan catabolite kynurenine to DC cultures in which IDO activity was blocked restored long-term IDO expression in wild-type DC but not in AhR-deficient DC, establishing the central role of the kynurenine-AhR pathway in maintaining IDO expression in tolerogenic DC. These findings shed further light on the cellular and molecular biology of the post-IL-12 regulatory rebound and provide insight into how feedback inhibitory mechanisms dominate in the long-term.

Regulatory Rebound in IL-12-Treated Tumors Is Driven by Uncommitted Peripheral Regulatory T Cells.

IL-12 promotes a rapid reversal of immune suppression in the tumor microenvironment. However, the adjuvant activity of IL-12 is short-lived due to regulatory T cell (Treg) reinfiltration. Quantitative analysis of Treg kinetics in IL-12-treated tumors and tumor-draining lymph nodes revealed a transient loss followed by a rapid 4-fold expansion of tumor Treg between days 3 and 10. Subset-specific analysis demonstrated that the posttreatment rebound was driven by the CD4(+)CD25(+)Foxp3(+) neuropilin-1(low) peripheral Treg (pTreg), resulting in a 3-5-fold increase in the pTreg to CD4(+)CD25(+)Foxp3(+) neuropilin-1(high) thymic Treg ratio by day 10. The expanding pTreg displayed hypermethylation of the CpG islands in Treg-specific demethylated region, CTLA-4 exon 2, and glucocorticoid-induced TNFR exon 5, were phenotypically unstable, and exhibited diminished suppressive function consistent with an uncommitted in vitro-induced Treg-like phenotype. In vitro culture of posttherapy Treg populations under Th1-promoting conditions resulted in higher levels of IFN-γ production by pTreg compared with thymic Treg, confirming their transitional state. Blockade of selected molecular mechanisms that are known to promote Treg expansion identified IDO-positive dendritic cells as the primary mediator of post-IL-12 pTreg expansion. Clinical implications of these findings are discussed.

Ontogeny of Tumor-associated CD4+CD25+Foxp3+ T-regulatory Cells.

The critical contribution of CD4+CD25+Foxp3+ T-regulatory cells (Treg) to immune suppression in the tumor microenvironment is well-established. Whereas the mechanisms that drive the generation and accumulation of Treg in tumors have been an active area of study, the information on their origin and population dynamics remains limited. In this review, we discuss the ontogeny of tumor-associated Treg in light of the recently identified lineage markers.

Radio-responsive tumors exhibit greater intratumoral immune activity than nonresponsive tumors.

Radiation therapy (RT) continues to be a cornerstone in the treatment for many cancers. Unfortunately, not all individuals respond effectively to RT resulting clinically in two groups consisting of nonresponders (progressive disease) and responders (tumor control/cure). The mechanisms that govern the outcome of radiotherapy are poorly understood. Interestingly, a new paradigm has emerged demonstrating that the immune system mediates many of the antitumor effects of RT. Therefore, we hypothesized that the immune response following RT may dictate the efficacy of treatment. To examine this, we developed a tumor model that mirrors this clinically relevant phenomenon in which mice bearing Colon38, a colon adenocarcinoma, were treated locally with 15Gy RT resulting in both nonresponders and responders. More importantly, we were able to distinguish responders from nonresponders as early as 4 days post-RT allowing for the unique opportunity to identify critical events that ultimately determined the effectiveness of therapy. Intratumoral immune cells and interferon-gamma were increased in responsive tumors and licensed CD8 T cells to exhibit lytic activity against tumor cells, a response that was diminished in tumors refractory to RT. Combinatorial treatment with RT and the immunomodulatory cytokine IL-12 resulted in complete remission of cancer in 100% of cases compared to a cure rate of only 12% with RT alone. Similar data were obtained when IL-12 was delivered by microspheres. Therefore, the efficacy of RT may depend on the strength of the immune response induced after radiotherapy. Additionally, immunotherapy that further stimulates the immune cells may enhance the effectiveness of RT.

Enhancement of adaptive immunity to Neisseria gonorrhoeae by local intravaginal administration of microencapsulated interleukin 12.

Gonorrhea remains one of the most frequent infectious diseases, and Neisseria gonorrhoeae is emerging as resistant to most available antibiotics, yet it does not induce a state of specific protective immunity against reinfection. Our recent studies have demonstrated that N. gonorrhoeae proactively suppresses host T-helper (Th) 1/Th2-mediated adaptive immune responses, which can be manipulated to generate protective immunity. Here we show that intravaginally administered interleukin 12 (IL-12) encapsulated in sustained-release polymer microspheres significantly enhanced both Th1 and humoral immune responses in a mouse model of genital gonococcal infection. Treatment of mice with IL-12 microspheres during gonococcal challenge led to faster clearance of infection and induced resistance to reinfection, with the generation of gonococcus-specific circulating immunoglobulin G and vaginal immunoglobulin A and G antibodies. These results suggest that local administration of microencapsulated IL-12 can serve as a novel therapeutic and prophylactic strategy against gonorrhea, with implications for the development of an effective vaccine.

Dichotomous effects of IFN-γ on dendritic cell function determine the extent of IL-12-driven antitumor T cell immunity.

Sustained intratumoral delivery of IL-12 and GM-CSF can overcome tumor immune suppression and promote T cell-dependent eradication of established disease in murine tumor models. However, the antitumor effector response is transient and rapidly followed by a T suppressor cell rebound. The mechanisms that control the switch from an effector to a regulatory response in this model have not been defined. Because dendritic cells (DC) can mediate both effector and suppressor T cell priming, DC activity was monitored in the tumors and the tumor-draining lymph nodes (TDLN) of IL-12/GM-CSF-treated mice. The studies demonstrated that therapy promoted the recruitment of immunogenic DC (iDC) to tumors with subsequent migration to the TDLN within 24-48 h of treatment. Longer-term monitoring revealed that iDC converted to an IDO-positive tolerogenic phenotype in the TDLN between days 2 and 7. Specifically, day 7 DC lost the ability to prime CD8(+) T cells but preferentially induced CD4(+)Foxp3(+) T cells. The functional switch was reversible, as inhibition of IDO with 1-methyl tryptophan restored immunogenic function to tolerogenic DC. All posttherapy immunological activity was strictly associated with conventional myeloid DC, and no functional changes were observed in the plasmacytoid DC subset throughout treatment. Importantly, the initial recruitment and activation of iDC as well as the subsequent switch to tolerogenic activity were both driven by IFN-γ, revealing the dichotomous role of this cytokine in regulating IL-12-mediated antitumor T cell immunity.

Chronic chemoimmunotherapy achieves cure of spontaneous murine mammary tumors via persistent blockade of posttherapy counter-regulation.

Intratumoral delivery of IL-12 and GM-CSF induces local and systemic antitumor CD8(+) T cell activation and tumor kill. However, the effector response is transient and is rapidly countered by CD4(+) Foxp3(+) T suppressor cell expansion. To determine whether depletion of the pre-existing T suppressor cell pool prior to treatment could diminish posttherapy regulatory cell resurgence, FVBneuN mice bearing advanced spontaneous mammary tumors were treated with cyclophosphamide (CY) 1 d before IL-12/GM-CSF therapy. Administration of CY mediated a significant delay in the post-IL-12/GM-CSF T suppressor cell rebound, resulting in a 7-fold increase in the CD8(+) CTL/T suppressor cell ratio, a 3-fold enhancement of CTL cytotoxicity, and an extension of the effector window from 3 to 7 d. In long-term therapy studies, chronic chemoimmunotherapy promoted a dramatic enhancement of tumor regression, resulting in complete cure in 44% of the mice receiving CY plus IL-12/GM-CSF. Tumor eradication in the chronic therapy setting was associated with the ability to repeatedly rescue and maintain cytotoxic CD8(+) T cell activity. These findings demonstrated that chronic administration of CY in conjunction with immune therapy enhances the initial induction of antitumor T effector cells and, more importantly, sustains their cytotoxic activity over the long-term via persistent blockade of homeostatic counter-regulation.

Microencapsulated IL-12 Drives Genital Tract Immune Responses to Intranasal Gonococcal Outer Membrane Vesicle Vaccine and Induces Resistance to Vaginal Infection with Diverse Strains of Neisseria gonorrhoeae.

An experimental gonococcal vaccine consisting of outer membrane vesicles (OMVs) and microsphere (ms)-encapsulated interleukin-12 (IL-12 ms) induces Th1-driven immunity, with circulating and genital antibodies to Neisseria gonorrhoeae, after intravaginal (i.vag.) administration in female mice, and generates resistance to vaginal challenge infection. Because i.vag. administration is inapplicable to males and may not be acceptable to women, we determined whether intranasal (i.n.) administration would generate protective immunity against N. gonorrhoeae. Female and male mice were immunized i.n. with gonococcal OMVs plus IL-12 ms or blank microspheres (blank ms). Responses to i.n. immunization were similar to those with i.vag. immunization, with serum IgG, salivary IgA, and vaginal IgG and IgA antigonococcal antibodies induced when OMVs were administered with IL-12 ms. Male mice responded with serum IgG and salivary IgA antibodies similarly to female mice. Gamma interferon (IFN-γ) production by CD4+ T cells from iliac lymph nodes was elevated after i.n. or i.vag. immunization with OMVs plus IL-12 ms. Female mice immunized with OMVs plus IL-12 ms by either route resisted challenge with N. gonorrhoeae to an equal extent, and resistance generated by i.n. immunization extended to heterologous strains of N. gonorrhoeae. Detergent-extracted OMVs, which have diminished lipooligosaccharide, generated protective immunity to challenge similar to native OMVs. OMVs from mutant N. gonorrhoeae, in which genes for Rmp and LpxL1 were deleted to eliminate the induction of blocking antibodies against Rmp and diminish lipooligosaccharide endotoxicity, also generated resistance to challenge infection similar to wild-type OMVs when administered i.n. with IL-12 ms. IMPORTANCE We previously demonstrated that female mice can be immunized intravaginally with gonococcal outer membrane vesicles (OMVs) plus microsphere (ms)-encapsulated interleukin-12 (IL-12 ms) to induce antigonococcal antibodies and resistance to genital tract challenge with live Neisseria gonorrhoeae. However, this route of vaccination may be impractical for human vaccine development and is inapplicable to males. Because intranasal immunization has previously been shown to induce antibody responses in both male and female genital tracts, we have evaluated this route of immunization with gonococcal OMVs plus IL-12 ms. In addition, we have refined the composition of gonococcal OMVs to reduce the endotoxicity of lipooligosaccharide and to eliminate the membrane protein Rmp, which induces countereffective blocking antibodies. The resulting vaccine may be more suitable for ultimate translation to human application against the sexually transmitted infection gonorrhea, which is becoming increasingly resistant to treatment with antibiotics.

Male microbiota-associated metabolite restores macrophage efferocytosis in female lupus-prone mice via activation of PPARγ/LXR signaling pathways.

Systemic lupus erythematosus development is influenced by both sex and the gut microbiota. Metabolite production is a major mechanism by which the gut microbiota influences the immune system, and we have previously found differences in the fecal metabolomic profiles of lupus-prone female and lupus-resistant male BWF1 mice. Here we determine how sex and microbiota metabolite production may interact to affect lupus. Transcriptomic analysis of female and male splenocytes showed genes that promote phagocytosis were upregulated in BWF1 male mice. Because patients with systemic lupus erythematosus exhibit defects in macrophage-mediated phagocytosis of apoptotic cells (efferocytosis), we compared splenic macrophage efferocytosis in vitro between female and male BWF1 mice. Macrophage efferocytosis was deficient in female compared to male BWF1 mice but could be restored by feeding male microbiota. Further transcriptomic analysis of the genes upregulated in male BWF1 mice revealed enrichment of genes stimulated by PPARγ and LXR signaling. Our previous fecal metabolomics analyses identified metabolites in male BWF1 mice that can activate PPARγ and LXR signaling and identified one in particular, phytanic acid, that is a very potent agonist. We show here that treatment of female BWF1 splenic macrophages with phytanic acid restores efferocytic activity via activation of the PPARγ and LXR signaling pathways. Furthermore, we found phytanic acid may restore female BWF1 macrophage efferocytosis through upregulation of the proefferocytic gene CD36. Taken together, our data indicate that metabolites produced by BWF1 male microbiota can enhance macrophage efferocytosis and, through this mechanism, could potentially influence lupus progression.

An extracellular vesicular mutant KRAS-associated protein complex promotes lung inflammation and tumor growth.

Extracellular vesicles (EVs) contain more than 100 proteins. Whether there are EVs proteins that act as an 'organiser' of protein networks to generate a new or different biological effect from that identified in EV-producing cells has never been demonstrated. Here, as a proof-of-concept, we demonstrate that EV-G12D-mutant KRAS serves as a leader that forms a protein complex and promotes lung inflammation and tumour growth via the Fn1/IL-17A/FGF21 axis. Mechanistically, in contrast to cytosol derived G12D-mutant KRAS complex from EVs-producing cells, EV-G12D-mutant KRAS interacts with a group of extracellular vesicular factors via fibronectin-1 (Fn1), which drives the activation of the IL-17A/FGF21 inflammation pathway in EV recipient cells. We show that: (i), depletion of EV-Fn1 leads to a reduction of a number of inflammatory cytokines including IL-17A; (ii) induction of IL-17A promotes lung inflammation, which in turn leads to IL-17A mediated induction of FGF21 in the lung; and (iii) EV-G12D-mutant KRAS complex mediated lung inflammation is abrogated in IL-17 receptor KO mice. These findings establish a new concept in EV function with potential implications for novel therapeutic interventions in EV-mediated disease processes.

Characterization of iNOS(+) Neutrophil-like ring cell in tumor-bearing mice.

BACKGROUND: Myeloid-derived Suppressor Cells (MDSC) have been identified as tumor-induced immature myeloid cells (IMC) with potent immune suppressive activity in cancer. Whereas strict phenotypic classification of MDSC has been challenging due to the highly heterogeneous nature of cell surface marker expression, use of functional markers such as Arginase and inducible nitric oxide synthase (iNOS) may represent a better categorization strategy. In this study we investigated whether iNOS could be utilized as a specific marker for the identification of a more informative homogenous MDSC subset. METHODS: Single-cell suspensions from tumors and other organs were prepared essentially by enzymatic digestion. Flow cytometric analysis was performed on a four-color flow cytometer. Morphology, intracellular structure and localization of iNOS(+) ring cells in the tumor were determined by cytospin analysis, immunofluorescence microscopy and immunohistochemistry, respectively. For functional analysis, iNOS(+) ring subset were sorted and tested in vitro cell culture experiments. Pharmacologic inhibition of iNOS was performed both in vivo and in vitro. RESULTS: The results showed that intracellular iNOS staining distinguished a granular iNOS(+) SSC(hi) CD11b(+) Gr-1(dim) F4/80(+) subset with ring-shaped nuclei (ring cells) among the CD11b(+) Gr-1(+) cell populations found in tumors. The intensity of the ring cell infiltrate correlated with tumor size and these cells constituted the second major tumor-infiltrating leukocyte subset found in established tumors. Although phenotypic analysis demonstrated that ring cells shared characteristics with tumor-associated macrophages (TAM), morphological analysis revealed a neutrophil-like appearance as detected by cytospin and immunofluorescence microscopy analysis. The presence of distinct iNOS filled granule-like structures located next to the cell membrane suggested that iNOS was stored in pre-formed vesicles and available for rapid release upon activation. Tumor biopsies showed large areas with infiltrating ring cells primarily surrounding necrotic areas. Importantly, these cells significantly impaired CD8(+) T-cell proliferation and induced apoptotic death. The intratumoral accumulation and suppressive activity of ring cells could be blocked through pharmacologic inhibition of iNOS, demonstrating the critical role of this enzyme in mediating both the differentiation and the activity of these cells. CONCLUSIONS: In this study, iNOS expression was linked to a homogeneous subset; ring cells with a particular phenotype and immune suppressive function, in a common and well-established murine tumor model; 4T-1. Since the absence of a Gr-1 homolog in humans has made the identification of MDSC much more challenging, use of iNOS as a functional marker of MDSC may also have clinical importance.

Indoleamine 2,3-dioxygenase and dendritic cell tolerogenicity.

This article summarizes the molecular and cellular mechanisms that regulate the activity of indoleamine 2,3-dioxygenase (IDO), a potent immune-suppressive enzyme, in dendritic cells (DCs). Specific attention is given to differential up-regulation of IDO in distinct DC subsets, its function in immune homeostasis/autoimmunity, infection and cancer; and the associated immunological outcomes. The review will conclude with a discussion of the poorly defined mechanisms that mediate the long-term maintenance of IDO-expression in response to inflammatory stimuli and how selective modulation of IDO activity may be used in the treatment of disease.