SATB1 Expression Governs Epigenetic Repression of PD-1 in Tumor-Reactive T Cells.

Despite the importance of programmed cell death-1 (PD-1) in inhibiting T cell effector activity, the mechanisms regulating its expression remain poorly defined. We found that the chromatin organizer special AT-rich sequence-binding protein-1 (Satb1) restrains PD-1 expression induced upon T cell activation by recruiting a nucleosome remodeling deacetylase (NuRD) complex to Pdcd1 regulatory regions. Satb1 deficienct T cells exhibited a 40-fold increase in PD-1 expression. Tumor-derived transforming growth factor ß (Tgf-ß) decreased Satb1 expression through binding of Smad proteins to the Satb1 promoter. Smad proteins also competed with the Satb1-NuRD complex for binding to Pdcd1 enhancers, releasing Pdcd1 expression from Satb1-mediated repression, Satb1-deficient tumor-reactive T cells lost effector activity more rapidly than wild-type lymphocytes at tumor beds expressing PD-1 ligand (CD274), and these differences were abrogated by sustained CD274 blockade. Our findings suggest that Satb1 functions to prevent premature T cell exhaustion by regulating Pdcd1 expression upon T cell activation. Dysregulation of this pathway in tumor-infiltrating T cells results in diminished anti-tumor immunity.

Phenotype and function of tumor-associated neutrophils and their subsets in early-stage human lung cancer.

Neutrophils accumulate in many types of human and murine tumors and represent a significant portion of tumor-infiltrating myeloid cells. Our current understanding of the role of neutrophils in tumor development has depended primarily on murine models of cancer. However, there are crucial species differences in the evolution of tumors, genetic diversity, immune and inflammatory responses, and intrinsic biology of neutrophils that might have a profound impact on the tumor development and function of neutrophils in mouse versus human tumors. To date, the majority of experimental approaches to study neutrophils in cancer patients have been limited to the examination of circulating blood neutrophils. The phenotype and function of tumor-associated neutrophils (TANs) in humans, particularly in the early stages of tumor development, have not been extensively investigated. Thus, the long-term goal of our work has been to characterize human TANs and determine their specific role in tumor development. Here, we summarize our findings on human TANs obtained from human early stage lung cancer patients. We will describe the phenotypes of different TAN subsets identified in early stage lung tumors, as well as their functional dialog with T cells.

Human Tumor-Associated Macrophages and Neutrophils Regulate Antitumor Antibody Efficacy through Lethal and Sublethal Trogocytosis.

The clinical benefits of tumor-targeting antibodies (tAb) are modest in solid human tumors. The efficacy of many tAbs is dependent on Fc receptor (FcR)-expressing leukocytes that bind Fc fragments of tAb. Tumor-associated macrophages (TAM) and neutrophils (TAN) represent the majority of FcR+ effectors in solid tumors. A better understanding of the mechanisms by which TAMs and TANs regulate tAb response could help improve the efficacy of cancer treatments. Here, we found that myeloid effectors interacting with tAb-opsonized lung cancer cells used antibody-dependent trogocytosis (ADT) but not antibody-dependent phagocytosis. During this process, myeloid cells "nibbled off" tumor cell fragments containing tAb/targeted antigen (tAg) complexes. ADT was only tumoricidal when the tumor cells expressed high levels of tAg and the effectors were present at high effector-to-tumor ratios. If either of these conditions were not met, which is typical for solid tumors, ADT was sublethal. Sublethal ADT, mainly mediated by CD32hiCD64hi TAM, led to two outcomes: (i) removal of surface tAg/tAb complexes from the tumor that facilitated tumor cell escape from the tumoricidal effects of tAb; and (ii) acquisition of bystander tAgs by TAM with subsequent cross-presentation and stimulation of tumor-specific T-cell responses. CD89hiCD32loCD64lo peripheral blood neutrophils (PBN) and TAN stimulated tumor cell growth in the presence of the IgG1 anti-EGFR Ab cetuximab; however, IgA anti-EGFR Abs triggered the tumoricidal activity of PBN and negated the stimulatory effect of TAN. Overall, this study provides insights into the mechanisms by which myeloid effectors mediate tumor cell killing or resistance during tAb therapy. SIGNIFICANCE: The elucidation of the conditions and mechanisms by which human FcR+ myeloid effectors mediate cancer cell resistance and killing during antibody treatment could help develop improved strategies for treating solid tumors.

Tumor factors stimulate lysosomal degradation of tumor antigens and undermine their cross-presentation in lung cancer.

Activities of dendritic cells (DCs) that present tumor antigens are often suppressed in tumors. Here we report that this suppression is induced by tumor microenvironment-derived factors, which activate the activating transcription factor-3 (ATF3) transcription factor and downregulate cholesterol 25-hydroxylase (CH25H). Loss of CH25H in antigen presenting cells isolated from human lung tumors is associated with tumor growth and lung cancer progression. Accordingly, mice lacking CH25H in DCs exhibit an accelerated tumor growth, decreased infiltration and impaired activation of intratumoral CD8+ T cells. These mice do not establish measurable long-term immunity against malignant cells that undergo chemotherapy-induced immunogenic cell death. Mechanistically, downregulation of CH25H stimulates membrane fusion between endo-phagosomes and lysosomes, accelerates lysosomal degradation and restricts cross-presentation of tumor antigens in the intratumoral DCs. Administration of STING agonist MSA-2 reduces the lysosomal activity in DCs, restores antigen cross presentation, and increases therapeutic efficacy of PD-1 blockade against tumour challenge in a CH25H-dependent manner. These studies highlight the importance of downregulation of CH25H in DCs for tumor immune evasion and resistance to therapy.

Folate Receptor Beta Designates Immunosuppressive Tumor-Associated Myeloid Cells That Can Be Reprogrammed with Folate-Targeted Drugs.

Although immunotherapies of tumors have demonstrated promise for altering the progression of malignancies, immunotherapies have been limited by an immunosuppressive tumor microenvironment (TME) that prevents infiltrating immune cells from performing their anticancer functions. Prominent among immunosuppressive cells are myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) that inhibit T cells via release of immunosuppressive cytokines and engagement of checkpoint receptors. Here, we explore the properties of MDSCs and TAMs from freshly isolated mouse and human tumors and find that an immunosuppressive subset of these cells can be distinguished from the nonimmunosuppressive population by its upregulation of folate receptor beta (FRß) within the TME and its restriction to the TME. This FRß+ subpopulation could be selectively targeted with folate-linked drugs. Delivery of a folate-targeted TLR7 agonist to these cells (i) reduced their immunosuppressive function, (ii) increased CD8+ T-cell infiltration, (iii) enhanced M1/M2 macrophage ratios, (iv) inhibited tumor growth, (v) blocked tumor metastasis, and (vi) improved overall survival without demonstrable toxicity. These data reveal a broadly applicable strategy across tumor types for reprogramming MDSCs and TAMs into antitumorigenic immune cells using a drug that would otherwise be too toxic to administer systemically. The data also establish FRß as the first marker that distinguishes immunosuppressive from nonimmunosuppressive subsets of MDSCs and TAMs. Because all solid tumors accumulate MDSCs and TAMs, a general strategy to both identify and reprogram these cells should be broadly applied in the characterization and treatment of multiple tumors. SIGNIFICANCE: FRß serves as both a means to identify and target MDSCs and TAMs within the tumor, allowing for delivery of immunomodulatory compounds to tumor myeloid cells in a variety of cancers.

Analysis of classical neutrophils and polymorphonuclear myeloid-derived suppressor cells in cancer patients and tumor-bearing mice.

In this study, using single-cell RNA-seq, cell mass spectrometry, flow cytometry, and functional analysis, we characterized the heterogeneity of polymorphonuclear neutrophils (PMNs) in cancer. We describe three populations of PMNs in tumor-bearing mice: classical PMNs, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), and activated PMN-MDSCs with potent immune suppressive activity. In spleens of mice, PMN-MDSCs gradually replaced PMNs during tumor progression. Activated PMN-MDSCs were found only in tumors, where they were present at the very early stages of the disease. These populations of PMNs in mice could be separated based on the expression of CD14. In peripheral blood of cancer patients, we identified two distinct populations of PMNs with characteristics of classical PMNs and PMN-MDSCs. The gene signature of tumor PMN-MDSCs was similar to that in mouse activated PMN-MDSCs and was closely associated with negative clinical outcome in cancer patients. Thus, we provide evidence that PMN-MDSCs are a distinct population of PMNs with unique features and potential for selective targeting opportunities.

ROS-producing immature neutrophils in giant cell arteritis are linked to vascular pathologies.

Giant cell arteritis (GCA) is a common form of primary systemic vasculitis in adults, with no reliable indicators of prognosis or treatment responses. We used single cell technologies to comprehensively map immune cell populations in the blood of patients with GCA and identified the CD66b+CD15+CD10lo/-CD64- band neutrophils and CD66bhiCD15+CD10lo/-CD64+/bright myelocytes/metamyelocytes to be unequivocally associated with both the clinical phenotype and response to treatment. Immature neutrophils were resistant to apoptosis, remained in the vasculature for a prolonged period of time, interacted with platelets, and extravasated into the tissue surrounding the temporal arteries of patients with GCA. We discovered that immature neutrophils generated high levels of extracellular reactive oxygen species, leading to enhanced protein oxidation and permeability of endothelial barrier in an in vitro coculture system. The same populations were also detected in other systemic vasculitides. These findings link functions of immature neutrophils to disease pathogenesis, establishing a clinical cellular signature of GCA and suggesting different therapeutic approaches in systemic vascular inflammation.

Lactate Limits T Cell Proliferation via the NAD(H) Redox State.

Immune cell function is influenced by metabolic conditions. Low-glucose, high-lactate environments, such as the placenta, gastrointestinal tract, and the tumor microenvironment, are immunosuppressive, especially for glycolysis-dependent effector T cells. We report that nicotinamide adenine dinucleotide (NAD+), which is reduced to NADH by lactate dehydrogenase in lactate-rich conditions, is a key point of metabolic control in T cells. Reduced NADH is not available for NAD+-dependent enzymatic reactions involving glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate dehydrogenase (PGDH). We show that increased lactate leads to a block at GAPDH and PGDH, leading to the depletion of post-GAPDH glycolytic intermediates, as well as the 3-phosphoglycerate derivative serine that is known to be important for T cell proliferation. Supplementing serine rescues the ability of T cells to proliferate in the presence of lactate-induced reductive stress. Directly targeting the redox state may be a useful approach for developing novel immunotherapies in cancer and therapeutic immunosuppression.

AAV3-mediated transfer and expression of the pyruvate dehydrogenase E1 alpha subunit gene causes metabolic remodeling and apoptosis of human liver cancer cells.

Most cancers rely disproportionately on glycolysis for energy even in the presence of adequate oxygen supply, a condition known as "aerobic glycolysis", or the Warburg effect. Pharmacological reversal of the Warburg effect has been shown to cause selective apoptosis of tumor cells, presumably by stimulating mitochondrial respiratory chain activity and production of reactive oxygen species that, in turn, induce a caspase-mediated series of reactions leading to cell death. We reasoned that a similar effect on tumor cells might result from up-regulation of the E1alpha subunit gene (pda1) of the pyruvate dehydrogenase complex (PDC) that catalyzes the rate-limiting step in aerobic glucose oxidation and thus plays a major role in the control of oxidative phosphorylation. To test this postulate, we employed a self-complementary adeno-associated virus (scAAV)-based delivery and expression system for targeting pda1 to the mitochondria of primary cultures of human hepatoblastoma (HB) and hepatocellular carcinoma (HCC) cells. Serotypes 1-10 scAAV vectors that included enhanced green fluorescent (egfp) reporter gene driven by either cytomegalovirus (CMV) or chicken beta-actin (CBA) promoters were analyzed for transduction ability of HB (Huh-6) and HCC (Huh-7 and HepG2) cell lines and primary cultures of normal human hepatocytes. Serotype 3 scAAV-egfp (scAAV3-egfp) vector was the most efficient and transduced up to 90% of cells. We limited the transgene expression primarily to liver cancer cells by generating scAAV3 vectors that contained the human alpha-fetoprotein promoter (AFP)-driven reporter gene (scAAV3.AFP-egfp) and the potentially therapeutic gene scAAV3.AFP-pda1. Infection of Huh-6 cells by the scAAV3.AFP-pda1 vector increased protein expression of E1alpha, PDC catalytic activity, and late-stage apoptotic cell death. Apoptosis was also associated with increased protein expression of Bcl-X/S, an early marker of apoptosis, and release of cytochrome c into the cytosol of infected HB cells. These data indicate that molecular targeting of mitochondrial oxidative metabolism in liver cancer cells by AAV3-mediated delivery of pda1 holds promise as a novel and effective therapeutic approach for human hepatic tumors.

Function of Human Tumor-Infiltrating Lymphocytes in Early-Stage Non-Small Cell Lung Cancer.

Cancer progression is marked by dysfunctional tumor-infiltrating lymphocytes (TIL) with high inhibitory receptor (IR) expression. Because IR blockade has led to clinical responses in some patients with non-small cell lung cancer (NSCLC), we investigated how IRs influenced CD8+ TIL function from freshly digested early-stage NSCLC tissues using a killing assay and intracellular cytokine staining after in vitro T-cell restimulation. Early-stage lung cancer TIL function was heterogeneous with only about one third of patients showing decrements in cytokine production and lytic function. TIL hypofunction did not correlate with clinical factors, coexisting immune cells (macrophages, neutrophils, or CD4+ T regulatory cells), nor with PD-1, TIGIT, TIM-3, CD39, or CTLA-4 expression. Instead, we found that the presence of the integrin αeß7 (CD103), characteristic of tissue-resident memory cells (TRM), was positively associated with cytokine production, whereas expression of the transcription factor Eomesodermin (Eomes) was negatively associated with TIL function. These data suggest that the functionality of CD8+ TILs from early-stage NSCLCs may be influenced by competition between an antitumor CD103+ TRM program and an exhaustion program marked by Eomes expression. Understanding the mechanisms of T-cell function in the progression of lung cancer may have clinical implications for immunotherapy.

Phenotypic and functional analysis of malignant mesothelioma tumor-infiltrating lymphocytes.

Given the growing interest and promising preliminary results of immunotherapy in malignant pleural mesothelioma (MPM), it has become important to more fully understand the immune landscape in this tumor. This may be especially relevant in deciding who might benefit most from checkpoint blockade or agonist antibody therapy. Since the phenotype of tumor infiltrating lymphocytes (TILs) in MPM has not been fully described and their function has not been carefully assessed, we collected fresh tumor and blood from 22 patients undergoing surgical resection and analysed single cell suspensions by flow cytometry. The functionality of TILs was assessed by measurement of cytokine expression (IFN-γ) following overnight stimulation ex vivo. Results showed low numbers of CD8+ TILs whose function was either moderately or severely suppressed. The degree of TIL hypofunction did not correlate with the presence of co-existing macrophages or neutrophils, nor with expression of the inhibitory receptors PD-1, CD39 and CTLA-4. Hypofunction was associated with higher numbers of CD4 regulatory T cells (Tregs) and with expression of the inhibitory receptor TIGIT. On the other hand, presence of tissue-resident memory (Trm) cells and expression of TIM-3 on CD8+ cells were positively associated with cytokine production. However, Trm function was partially suppressed when the transcription factor Eomesodermin (Eomes) was co-expressed. Understanding the function of TILs in malignant mesothelioma may have clinical implications for immunotherapy, especially in choosing the best immunotherapy targets. Our data suggests that Treg cell blocking agents or TIGIT inhibitor antibodies might be especially valuable in these patients.

Human tumor-associated monocytes/macrophages and their regulation of T cell responses in early-stage lung cancer.

Data from mouse tumor models suggest that tumor-associated monocyte/macrophage lineage cells (MMLCs) dampen antitumor immune responses. However, given the fundamental differences between mice and humans in tumor evolution, genetic heterogeneity, and immunity, the function of MMLCs might be different in human tumors, especially during early stages of disease. Here, we studied MMLCs in early-stage human lung tumors and found that they consist of a mixture of classical tissue monocytes and tumor-associated macrophages (TAMs). The TAMs coexpressed M1/M2 markers, as well as T cell coinhibitory and costimulatory receptors. Functionally, TAMs did not primarily suppress tumor-specific effector T cell responses, whereas tumor monocytes tended to be more T cell inhibitory. TAMs expressing relevant MHC class I/tumor peptide complexes were able to activate cognate effector T cells. Mechanistically, programmed death-ligand 1 (PD-L1) expressed on bystander TAMs, as opposed to PD-L1 expressed on tumor cells, did not inhibit interactions between tumor-specific T cells and tumor targets. TAM-derived PD-L1 exerted a regulatory role only during the interaction of TAMs presenting relevant peptides with cognate effector T cells and thus may limit excessive activation of T cells and protect TAMs from killing by these T cells. These results suggest that the function of TAMs as primarily immunosuppressive cells might not fully apply to early-stage human lung cancer and might explain why some patients with strong PD-L1 positivity fail to respond to PD-L1 therapy.

Mouse versus Human Neutrophils in Cancer: A Major Knowledge Gap.

Many types of cancer recruit neutrophils that could have protumor or antitumor effects on tumor development. Numerous findings in murine models suggest a predominantly protumoral role for neutrophils in cancer development. However, there are fundamental differences between mouse and human tumors in the evolution of tumors, genetic diversity, immune response, and also in the intrinsic biology of neutrophils that might have a profound impact on tumor development and the function of these cells. A crucial difference is that the majority of mouse tumor models lack the prolonged initial phases of multistage tumor evolution present in humans when antitumoral mechanisms are activated. In this review, we discuss the challenges specific to cross-species extrapolation of neutrophil function during mouse versus human tumor development.

Human lung tumor FOXP3+ Tregs upregulate four "Treg-locking" transcription factors.

Experimental data indicate that FOXP3+ Tregs can markedly curtail host antitumor immune responses, but the properties of human intratumoral Tregs are still largely unknown, in part due to significant methodologic problems. We studied the phenotypic, functional, epigenetic, and transcriptional features of Tregs in 92 patients with non-small-cell lung cancer, comparing the features of Tregs within tumors versus corresponding blood, lung, and lymph node samples. Intratumoral Treg numbers and suppressive function were significantly increased compared with all other sites but did not display a distinctive phenotype by flow cytometry. However, by undertaking simultaneous evaluation of mRNA and protein expression at the single-cell level, we demonstrated that tumor Tregs have a phenotype characterized by upregulated expression of FOXP3 mRNA and protein as well as significantly increased expression of EOS, IRF4, SATB1, and GATA1 transcription factor mRNAs. Expression of these "Treg-locking" transcription factors was positively correlated with levels of FOXP3 mRNA, with highest correlations for EOS and SATB1. EOS had an additional, FOXP3 mRNA-independent, positive correlation with FOXP3 protein in tumor Tregs. Our study identifies distinctive features of intratumoral Tregs and suggests that targeting Treg-locking transcription factors, especially EOS, may be of clinical importance for antitumor Treg-based therapy.

Tumor Cell-Independent Estrogen Signaling Drives Disease Progression through Mobilization of Myeloid-Derived Suppressor Cells.

The role of estrogens in antitumor immunity remains poorly understood. Here, we show that estrogen signaling accelerates the progression of different estrogen-insensitive tumor models by contributing to deregulated myelopoiesis by both driving the mobilization of myeloid-derived suppressor cells (MDSC) and enhancing their intrinsic immunosuppressive activity in vivo Differences in tumor growth are dependent on blunted antitumor immunity and, correspondingly, disappear in immunodeficient hosts and upon MDSC depletion. Mechanistically, estrogen receptor alpha activates the STAT3 pathway in human and mouse bone marrow myeloid precursors by enhancing JAK2 and SRC activity. Therefore, estrogen signaling is a crucial mechanism underlying pathologic myelopoiesis in cancer. Our work suggests that new antiestrogen drugs that have no agonistic effects may have benefits in a wide range of cancers, independently of the expression of estrogen receptors in tumor cells, and may synergize with immunotherapies to significantly extend survival. SIGNIFICANCE: Ablating estrogenic activity delays malignant progression independently of the tumor cell responsiveness, owing to a decrease in the mobilization and immunosuppressive activity of MDSCs, which boosts T-cell-dependent antitumor immunity. Our results provide a mechanistic rationale to block estrogen signaling with newer antagonists to boost the effectiveness of anticancer immunotherapies. Cancer Discov; 7(1); 72-85. ©2016 AACR.See related commentary by Welte et al., p. 17This article is highlighted in the In This Issue feature, p. 1.

Antigen-Presenting Intratumoral B Cells Affect CD4+ TIL Phenotypes in Non-Small Cell Lung Cancer Patients.

Effective immunotherapy options for patients with non-small cell lung cancer (NSCLC) are becoming increasingly available. The immunotherapy focus has been on tumor-infiltrating T cells (TILs); however, tumor-infiltrating B cells (TIL-Bs) have also been reported to correlate with NSCLC patient survival. The function of TIL-Bs in human cancer has been understudied, with little focus on their role as antigen-presenting cells and their influence on CD4+ TILs. Compared with other immune subsets detected in freshly isolated primary tumors from NSCLC patients, we observed increased numbers of intratumoral B cells relative to B cells from tumor-adjacent tissues. Furthermore, we demonstrated that TIL-Bs can efficiently present antigen to CD4+ TILs and alter the CD4+ TIL phenotype using an in vitro antigen-presentation assay. Specifically, we identified three CD4+ TIL responses to TIL-Bs, which we categorized as activated, antigen-associated, and nonresponsive. Within the activated and antigen-associated CD4+ TIL population, activated TIL-Bs (CD19+CD20+CD69+CD27+CD21+) were associated with an effector T-cell response (IFNγ+ CD4+ TILs). Alternatively, exhausted TIL-Bs (CD19+CD20+CD69+CD27-CD21-) were associated with a regulatory T-cell phenotype (FoxP3+ CD4+ TILs). Our results demonstrate a new role for TIL-Bs in NSCLC tumors in their interplay with CD4+ TILs in the tumor microenvironment, establishing them as a potential therapeutic target in NSCLC immunotherapy. Cancer Immunol Res; 5(10); 898-907. ©2017 AACR.

Trametinib Drives T-cell-Dependent Control of KRAS-Mutated Tumors by Inhibiting Pathological Myelopoiesis.

Targeted therapies elicit seemingly paradoxical and poorly understood effects on tumor immunity. Here, we show that the MEK inhibitor trametinib abrogates cytokine-driven expansion of monocytic myeloid-derived suppressor cells (mMDSC) from human or mouse myeloid progenitors. MEK inhibition also reduced the production of the mMDSC chemotactic factor osteopontin by tumor cells. Together, these effects reduced mMDSC accumulation in tumor-bearing hosts, limiting the outgrowth of KRas-driven breast tumors, even though trametinib largely failed to directly inhibit tumor cell proliferation. Accordingly, trametinib impeded tumor progression in vivo through a mechanism requiring CD8+ T cells, which was paradoxical given the drug's reported ability to inhibit effector lymphocytes. Confirming our observations, adoptive transfer of tumor-derived mMDSC reversed the ability of trametinib to control tumor growth. Overall, our work showed how the effects of trametinib on immune cells could partly explain its effectiveness, distinct from its activity on tumor cells themselves. More broadly, by providing a more incisive view into how MEK inhibitors may act against tumors, our findings expand their potential uses to generally block mMDSC expansion, which occurs widely in cancers to drive their growth and progression. Cancer Res; 76(21); 6253-65. ©2016 AACR.

Origin and Role of a Subset of Tumor-Associated Neutrophils with Antigen-Presenting Cell Features in Early-Stage Human Lung Cancer.

Based on studies in mouse tumor models, granulocytes appear to play a tumor-promoting role. However, there are limited data about the phenotype and function of tumor-associated neutrophils (TANs) in humans. Here, we identify a subset of TANs that exhibited characteristics of both neutrophils and antigen-presenting cells (APCs) in early-stage human lung cancer. These APC-like "hybrid neutrophils," which originate from CD11b(+)CD15(hi)CD10(-)CD16(low) immature progenitors, are able to cross-present antigens, as well as trigger and augment anti-tumor T cell responses. Interferon-γ and granulocyte-macrophage colony-stimulating factor are requisite factors in the tumor that, working through the Ikaros transcription factor, synergistically exert their APC-promoting effects on the progenitors. Overall, these data demonstrate the existence of a specialized TAN subset with anti-tumor capabilities in human cancer.

Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer.

Infiltrating inflammatory cells are highly prevalent within the tumor microenvironment and mediate many processes associated with tumor progression; however, the contribution of specific populations remains unclear. For example, the nature and function of tumor-associated neutrophils (TANs) in the cancer microenvironment is largely unknown. The goal of this study was to provide a phenotypic and functional characterization of TANs in surgically resected lung cancer patients. We found that TANs constituted 5%-25% of cells isolated from the digested human lung tumors. Compared with blood neutrophils, TANs displayed an activated phenotype (CD62L(lo)CD54(hi)) with a distinct repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. TANs produced substantial quantities of the proinflammatory factors MCP-1, IL-8, MIP-1α, and IL-6, as well as the antiinflammatory IL-1R antagonist. Functionally, both TANs and neutrophils isolated from distant nonmalignant lung tissue were able to stimulate T cell proliferation and IFN-γ release. Cross-talk between TANs and activated T cells led to substantial upregulation of CD54, CD86, OX40L, and 4-1BBL costimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive-feedback loop. Together our results demonstrate that in the earliest stages of lung cancer, TANs are not immunosuppressive, but rather stimulate T cell responses.

Progression of pulmonary tuberculosis and efficiency of bacillus Calmette-Guérin vaccination are genetically controlled via a common sst1-mediated mechanism of innate immunity.

Using a mouse model for genetic analysis of host resistance to virulent Mycobacterium tuberculosis, we have identified a genetic locus sst1 on mouse chromosome 1, which controls progression of pulmonary tuberculosis. In vitro, this locus had an effect on macrophage-mediated control of two intracellular bacterial pathogens, M. tuberculosis and Listeria monocytogenes. In this report, we investigated a specific function of the sst1 locus in antituberculosis immunity in vivo, especially its role in control of pulmonary tuberculosis. We found that the sst1 locus affected neither activation of Th1 cytokine-producing T lymphocytes, nor their migration to the lungs, but rather controlled an inducible NO synthase-independent mechanism of innate immunity. Although the sst1(S) macrophages responded to stimulation with IFN-gamma in vitro, their responsiveness to activation by T cells was impaired. Boosting T cell-mediated immunity by live attenuated vaccine Mycobacterium bovis bacillus Calmette-Guérin or the adoptive transfer of mycobacteria-activated CD4(+) T lymphocytes had positive systemic effect, but failed to improve control of tuberculosis infection specifically in the lungs of the sst1(S) animals. Thus, in the mouse model of tuberculosis, a common genetic mechanism of innate immunity mediated control of tuberculosis progression in the lungs and the efficiency of antituberculosis vaccine. Our data suggest that in immunocompetent humans the development of pulmonary tuberculosis and the failure of the existing vaccine to protect against it, in some cases, may be explained by a similar defect in a conserved inducible NO synthase-independent mechanism of innate immunity, either inherited or acquired.