Phase II Clinical Trial of Pembrolizumab and Chemotherapy Reveals Distinct Transcriptomic Profiles by Radiologic Response in Metastatic Triple-Negative Breast Cancer.

PURPOSE: A single arm, phase II trial of carboplatin, nab-paclitaxel, and pembrolizumab (CNP) in metastatic triple-negative breast cancer (mTNBC) was designed to evaluate overall response rate (ORR), progression-free survival (PFS), duration of response (DOR), safety/tolerability, overall survival (OS), and identify pathologic and transcriptomic correlates of response to therapy. PATIENTS AND METHODS: Patients with ≤2 prior therapies for metastatic disease were treated with CNP regardless of tumor programmed cell death-ligand 1 status. Core tissue biopsies were obtained prior to treatment initiation. ORR was assessed using a binomial distribution. Survival was analyzed via the Kaplan-Meier method. Bulk RNA sequencing was employed for correlative studies. RESULTS: Thirty patients were enrolled. The ORR was 48.0%: 2 (7%) complete responses (CR), 11 (41%) partial responses (PR), and 8 (30%) stable disease (SD). The median DOR for patients with CR or PR was 6.4 months [95% confidence interval (CI), 4-8.5 months]. For patients with CR, DOR was >24 months. Overall median PFS and OS were 5.8 (95% CI, 4.7-8.5 months) and 13.4 months (8.9-17.3 months), respectively. We identified unique transcriptomic landscapes associated with each RECIST category of radiographic treatment response. In CR and durable PR, IGHG1 expression was enriched. IGHG1high tumors were associated with improved OS (P = 0.045) and were concurrently enriched with B cells and follicular helper T cells, indicating IGHG1 as a promising marker for lymphocytic infiltration and robust response to chemo-immunotherapy. CONCLUSIONS: Pretreatment tissue sampling in mTNBC treated with CNP reveals transcriptomic signatures that may predict radiographic responses to chemo-immunotherapy.

Immunotherapy with IL12 and PD1/CTLA4 inhibition is effective in advanced ovarian cancer and associates with reversal of myeloid cell-induced immunosuppression.

The tumor microenvironment (TME) in ovarian cancer (OC) is characterized by immune suppression, due to an abundance of suppressive immune cells populations. To effectively enhance the activity of immune checkpoint inhibition (ICI), there is a need to identify agents that target these immunosuppressive networks while promoting the recruitment of effector T cells into the TME. To this end, we sought to investigate the effect of the immunomodulatory cytokine IL12 alone or in combination with dual-ICI (anti-PD1 + anti-CTLA4) on anti-tumor activity and survival, using the immunocompetent ID8-VEGF murine OC model. Detailed immunophenotyping of peripheral blood, ascites, and tumors revealed that durable treatment responses were associated with reversal of myeloid cell-induced immune suppression, which resulted in enhanced anti-tumor activity by T cells. Single cell transcriptomic analysis further demonstrated striking differences in the phenotype of myeloid cells from mice treated with IL12 in combination with dual-ICI. We also identified marked differences in treated mice that were in remission compared to those whose tumors progressed, further confirming a pivotal role for the modulation of myeloid cell function to allow for response to immunotherapy. These findings provide the scientific basis for the combination of IL12 and ICI to improve clinical response in OC.

Myeloid-cell-specific role of Gasdermin D in promoting lung cancer progression in mice.

The activities of the NLRP3 and AIM2 inflammasomes and Gasdermin D (GsdmD) are implicated in lung cancer pathophysiology but it's not clear if their contributions promote or retard lung cancer progression. Using a metastatic Lewis lung carcinoma (LLC) cell model, we show that GsdmD knockout (GsdmD-/-) mice form significantly fewer cancer foci in lungs, exhibit markedly decreased lung cancer metastasis, and show a significant ∼50% increase in median survival rate. The cleaved forms of GsdmD and IL-1ß were detected in lung tumor tissue, indicating inflammasome activity in lung tumor microenvironment (TME). Increased migration and growth of LLC cells was observed upon exposure to the conditioned media derived from inflammasome-induced wild type, but not the GsdmD-/-, macrophages. Using bone marrow transplantations, we show a myeloid-specific contribution of GsdmD in lung cancer metastasis. Taken together, our data show that GsdmD plays a myeloid-specific role in lung cancer progression.

Old is new again: emergence of thromboembolic complications in cancer patients on immunotherapy.

Cancer immunotherapy has emerged as one of the most important new treatments for cancer in many years, moving rapidly to front-line therapy for several cancers. Cancer immunotherapy is based on treatment with immune checkpoint inhibitors (ICI), which are monoclonal antibodies directed toward immunoregulatory proteins including PD-1, PD-L1 and CTLA-4. ICI inhibit interactions between these proteins and their ligands, disabling physiologic immune regulatory networks and enhancing anti-tumor immunity. However, since the immune response cannot be directed specifically to the tumor, ICI are associated with immune-related adverse events (irAEs) resulting from immune-mediated attack of normal tissues. We and others have reported a high incidence of thrombosis in patients treated with ICI, which may approach 20%. Given the rapidly increasing use of ICIs, it is clear that ICI-Associated Thrombosis (IAT) is a major emerging clinical problem. However, there is a remarkable knowledge gap concerning mechanisms of IAT. IAT may be a composite irAE resulting from activation of blood and vascular cells, leading to thromboinflammation. Cancer itself is an inflammatory disorder, and inducing further inflammation through ICI administration may stimulate procoagulant activity by multiple cell types. Moreover, some blood and vascular cells express ICI target proteins. Here, we review the results of several studies describing the clinical manifestations of IAT, as well as our recent studies demonstrating that elevated levels of myeloid derived suppressor cells and inflammatory cytokines may serve as biomarkers of IAT. It is hoped that the concepts reviewed here may stimulate further research into this important clinical problem.

Polyphosphate expression by cancer cell extracellular vesicles mediates binding of factor XII and contact activation.

Extracellular vesicles (EV) have been implicated in diverse biological processes, including intracellular communication, transport of nucleic acids, and regulation of vascular function. Levels of EVs are elevated in cancer, and studies suggest that EV may stimulate thrombosis in patients with cancer through expression of tissue factor. However, limited data also implicate EV in the activation of the contact pathway of coagulation through activation of factor XII (FXII) to FXIIa. To better define the ability of EV to initiate contact activation, we compared the ability of EV derived from different cancer cell lines to activate FXII. EV from all cell lines activated FXII, with those derived from pancreatic and lung cancer cell lines demonstrating the most potent activity. Concordant with the activation of FXII, EV induced the cleavage of high molecular weight kininogen (HK) to cleaved kininogen. We also observed that EVs from patients with cancer stimulated FXII activation and HK cleavage. To define the mechanisms of FXII activation by EV, EV were treated with calf intestinal alkaline phosphatase or Escherichia coli exopolyphosphatase to degrade polyphosphate; this treatment blocked binding of FXII to EVs and the ability of EV to mediate FXII activation. In vivo, EV induced pulmonary thrombosis in wild-type mice, with protection conferred by a deficiency in FXII, HK, or prekallikrein. Moreover, pretreatment of EVs with calf intestinal alkaline phosphatase inhibited their prothrombotic effect. These results indicate that polyphosphate mediates the binding of contact factors to EV and that EV-associated polyphosphate may contribute to the prothrombotic effects of EV in cancer.

Increased Incidence of Venous Thromboembolism with Cancer Immunotherapy.

BACKGROUND: Cancer immunotherapy is associated with several immune-related adverse events, but the relationship between immunotherapy and venous thromboembolism has not been thoroughly studied. METHODS: We conducted a retrospective cohort study of 1,686 patients who received immunotherapy for a variety of malignancies to determine the incidence of venous thromboembolism and the impact of venous thromboembolism on survival. To examine the potential role of inflammation in venous thromboembolism, we also profiled immune cells and plasma cytokines in blood samples obtained prior to initiation of immunotherapy in a sub-cohort of patients treated on clinical trials who subsequently did (N = 15), or did not (N = 10) develop venous thromboembolism. FINDINGS: Venous thromboembolism occurred while on immunotherapy in 404/1686 patients (24%) and was associated with decreased overall survival [HR=1.22 (95% CI 1.06-1.41), p<0.008]. Patients that developed venous thromboembolism had significantly higher pretreatment levels of myeloid-derived suppressor cells (5.382 ± 0.873 vs. 3.341 ± 0.3402, mean ± SEM; p=0.0045), interleukin 8 (221.2 ± 37.53 vs. 111.6 ± 25.36, mean ± SEM; p=0.016), and soluble vascular cell adhesion protein 1 (1210 ± 120.6 vs. 895.5 ± 53.34, mean ± SEM; p=0.0385). CONCLUSIONS: These findings demonstrate that venous thromboembolism is an underappreciated and important immune-related adverse event associated with cancer immunotherapy, and may implicate an interleukin 8 and myeloid-derived suppressor cell-driven pathway in pathogenesis.

Epithelial-derived gasdermin D mediates nonlytic IL-1ß release during experimental colitis.

Gasdermin D (GSDMD) induces pyroptosis via the pore-forming activity of its N-terminal domain, cleaved by activated caspases associated with the release of IL-1ß. Here, we report a nonpyroptotic role of full-length GSDMD in guiding the release of IL-1ß-containing small extracellular vesicles (sEVs) from intestinal epithelial cells (IECs). In response to caspase-8 inflammasome activation, GSDMD, chaperoned by Cdc37/Hsp90, recruits the E3 ligase, NEDD4, to catalyze polyubiquitination of pro-IL-1ß, serving as a signal for cargo loading into secretory vesicles. GSDMD and IL-1ß colocalize with the exosome markers CD63 and ALIX intracellularly, and GSDMD and NEDD4 are required for release of CD63+ sEVs containing IL-1ß, GSDMD, NEDD4, and caspase-8. Importantly, increased expression of epithelial-derived GSDMD is observed both in patients with inflammatory bowel disease (IBD) and those with experimental colitis. While GSDMD-dependent release of IL-1ß-containing sEVs is detected in cultured colonic explants from colitic mice, GSDMD deficiency substantially attenuates disease severity, implicating GSDMD-mediated release of IL-1ß sEVs in the pathogenesis of intestinal inflammation, such as that observed in IBD.

TRAF Regulation of IL-17 Cytokine Signaling.

Tumor necrosis factor receptor (TNFR)-associated factors or (TRAFs) are important mediators of Interleukin-17 (IL-17) cytokine signaling and contribute to driving tissue responses that are crucial for protective immunity but are often implicated in immunopathology. By amplifying tissue immune activity, IL-17 cytokine pathways contribute to maintaining barrier function as well as activation of innate and adaptive immunity necessary for host defense. IL-17 receptors signaling is orchestrated in part, by the engagement of TRAFs and the subsequent unlocking of downstream cellular machinery that can promote pathogen clearance or contribute to immune dysregulation, chronic inflammation, and disease. Originally identified as signaling adaptors for TNFR superfamily, TRAF proteins can mediate the signaling of a variety of intercellular and extracellular stimuli and have been shown to regulate the downstream activity of many cytokine receptors including receptors for IL-1ß, IL-2, IL-6, IL-17, IL-18, IL-33, type I IFNs, type III IFNs, GM-CSF, M-CSF, and TGF-ß Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I- like receptors, and C-type lectin receptors. This review will focus on discussing studies that reveal our current understanding of how TRAFs mediate and regulate biochemical activities downstream of the IL-17 cytokines signaling.

Hyaluronan Rafts on Airway Epithelial Cells.

Many cells, including murine airway epithelial cells, respond to a variety of inflammatory stimuli by synthesizing leukocyte-adhesive hyaluronan (HA) cables that remain attached to their cell surfaces. This study shows that air-liquid interface cultures of murine airway epithelial cells (AECs) also actively synthesize and release a majority of their HA onto their ciliated apical surfaces to form a heavy chain hyaluronan (HC-HA) matrix in the absence of inflammatory stimuli. These matrices do not resemble the rope-like HA cables but occur in distinct sheets or rafts that can capture and embed leukocytes from cell suspensions. The HC-HA modification involves the transfer of heavy chains from the inter-α-inhibitor (IαI) proteoglycan, which has two heavy chains (HC1 and HC2) on its chondroitin sulfate chain. The transesterification transfer of HCs from chondroitin sulfate to HA is mediated by tumor necrosis factor-induced gene 6 (TSG-6), which is up-regulated in inflammatory reactions. Because the AEC cultures do not have TSG-6 nor serum, the source of IαI, assays for HCs and TSG-6 were done. The results show that AECs synthesize TSG-6 and their own heavy chain donor (pre-IαI) with a single heavy chain 3 (HC3), which are also constitutively expressed by human renal proximal tubular epithelial cells. These leukocyte adhesive HC3-HA structures were also found in the bronchoalveolar lavage of naïve mice and were observed on their apical ciliated surfaces. Thus, these leukocyte-adhesive HA rafts are now identified as HC3-HA complexes that could be part of a host defense mechanism filling some important gaps in our current understanding of murine airway epithelial biology and secretions.

TSG-6 protein is crucial for the development of pulmonary hyaluronan deposition, eosinophilia, and airway hyperresponsiveness in a murine model of asthma.

Hyaluronan (HA) deposition is often correlated with mucosal inflammatory responses, where HA mediates both protective and pathological responses. By modifying the HA matrix, Tnfip6 (TNF-α-induced protein-6; also known as TSG-6 (TNF-stimulated gene-6)) is thought to potentiate anti-inflammatory and anti-plasmin effects that are inhibitory to leukocyte extravasation. In this study, we examined the role of endogenous TSG-6 in the pathophysiological responses associated with acute allergic pulmonary inflammation. Compared with wild-type littermate controls, TSG-6(-/-) mice exhibited attenuated inflammation marked by a significant decrease in pulmonary HA concentrations measured in the bronchoalveolar lavage and lung tissue. Interestingly, despite the equivalent induction of both humoral and cellular Th2 immunity and the comparable levels of cytokines and chemokines typically associated with eosinophilic pulmonary inflammation, airway eosinophilia was significantly decreased in TSG-6(-/-) mice. Most importantly, contrary to their counterpart wild-type littermates, TSG-6(-/-) mice were resistant to the induction of airway hyperresponsiveness and manifested improved lung mechanics in response to methacholine challenge. Our study demonstrates that endogenous TSG-6 is dispensable for the induction of Th2 immunity but is essential for the robust increase in pulmonary HA deposition, propagation of acute eosinophilic pulmonary inflammation, and development of airway hyperresponsiveness. Thus, TSG-6 is implicated in the experimental murine model of allergic pulmonary inflammation and is likely to contribute to the pathogenesis of asthma.

Tumor necrosis factor-stimulated gene-6 (TSG-6) amplifies hyaluronan synthesis by airway smooth muscle cells.

We tested the hypothesis that the artificial addition of heavy chains from inter-α-inhibitor to hyaluronan (HA), by adding recombinant TSG-6 (TNF-stimulated gene-6) to the culture medium of murine airway smooth muscle (MASM) cells, would enhance leukocyte binding to HA cables produced in response to poly(I:C). As predicted, the addition of heavy chains to HA cables enhanced leukocyte adhesion to these cables, but it also had several unexpected effects. (i) It produced thicker, more pronounced HA cables. (ii) It increased the accumulation of HA in the cell-associated matrix. (iii) It decreased the amount of HA in the conditioned medium. Importantly, these effects were observed only when TSG-6 was administered in the presence of poly(I:C), and TSG-6 did not exert any effect on its own. Increased HA synthesis occurred during active, poly(I:C)-induced HA synthesis and did not occur when TSG-6 was added after poly(I:C)-induced HA synthesis was complete. MASM cells derived from TSG-6(-/-), HAS1/3(-/-), and CD44(-/-) mice amplified HA synthesis in response to poly(I:C) + TSG-6 in a manner similar to WT MASM cells, demonstrating that they are expendable in this process. We conclude that TSG-6 increases the accumulation of HA in the cell-associated matrix, partially by preventing its dissolution from the cell-associated matrix into the conditioned medium, but primarily by inducing HA synthesis.

Correlation of hyaluronan deposition with infiltration of eosinophils and lymphocytes in a cockroach-induced murine model of asthma.

Asthma is a chronic inflammatory disease that exhibits airway remodeling with changes in the extracellular matrix (ECM). The role of the ECM in mediating these changes is poorly understood. Hyaluronan (HA), a major component of the ECM, has been implicated in many biological processes in diseases. This study investigates the processes involved in HA synthesis, deposition and localization during the propagation of cockroach-induced asthma. Mice were sensitized and challenged with cockroach antigen, and sacrificed at various time points during an 8-week challenge protocol. Analysis of bronchoalveolar lavage (BAL) fluid revealed an increase in total nucleated cells as early as 6h, which peaked at 6 days. Histopathologic analysis of the lung tissue revealed an influx of inflammatory cells at the peribronchial and perivascular regions starting at 12 h, which peaked at 6 days and persisted to 8 weeks. Eosinophils predominated in the early time points while lymphocytes predominated during the late time points. Quantitative polymerase chain reaction (PCR) data showed that hyaluronan synthase 1 (HAS1) mRNA peaked within 6 h and then declined. HAS2 mRNA also peaked within 6 h but remained elevated throughout the 8-week exposure course. HA levels in lung tissue and BAL increased at 12 h and peaked by 6 and 8 days, respectively. Inflammatory cells and new collagen formation localized in areas of HA deposition. Taken together, these data support a role for HA in the pathogenesis in asthma.

Epithelial cell-specific Act1 adaptor mediates interleukin-25-dependent helminth expulsion through expansion of Lin(-)c-Kit(+) innate cell population.

Interleukin-25 (IL-25 or IL-17E), a member of the structurally related IL-17 family, functions as an important mediator of T helper 2 cell-type (type 2) responses. We examined the cell type-specific role of IL-25-induced Act1-mediated signaling in protective immunity against helminth infection. Targeted Act1 deficiency in epithelial cells resulted in a marked delay in worm expulsion and abolished the expansion of the Lin(-)c-Kit(+) innate cell population in the mesenteric lymph node, lung, and liver. Th2 cell-inducing cytokine (IL-25 and IL-33) expression were reduced in the intestinal epithelial cells from the infected and IL-25-injected epithelial-specific Act1-deficient mice. Adoptive transfer of Lin(-)c-Kit(+) cells or combined injection of IL-25 and IL-33 restored the type 2 responses in these mice. Taken together, these results suggest that epithelial-specific Act1 mediates the expansion of the Lin(-)c-Kit(+) innate cell population through the positive-feedback loop of IL-25, initiating the type 2 immunity against helminth infection.

A CC' loop decoy peptide blocks the interaction between Act1 and IL-17RA to attenuate IL-17- and IL-25-induced inflammation.

Interleukin-17 (IL-17) and IL-25 signaling induce the expression of genes encoding inflammatory factors and are implicated in the pathology of various inflammatory diseases. Nuclear factor κB (NF-κB) activator 1 (Act1) is an adaptor protein and E3 ubiquitin ligase that is critical for signaling by either IL-17 or IL-25, and it is recruited to their receptors (IL-17R and IL-25R) through heterotypic interactions between the SEFIR [SEF (similar expression to fibroblast growth factor genes) and IL-17R] domain of Act1 and that of the receptor. SEFIR domains have structural similarity with the Toll-IL-1 receptor (TIR) domains of Toll-like receptors and IL-1R. Whereas the BB' loop of TIR is required for TIR-TIR interactions, we found that deletion of the BB' loop from Act1 or IL-17RA (a common subunit of both IL-17R and IL-25R) did not affect Act1-IL-17RA interactions; rather, deletion of the CC' loop from Act1 or IL-17RA abolished the interaction between both proteins. Surface plasmon resonance measurements showed that a peptide corresponding to the CC' loop of Act1 bound directly to IL-17RA. A cell-permeable decoy peptide based on the CC' loop sequence inhibited IL-17- or IL-25-mediated signaling in vitro, as well as IL-17- and IL-25-induced pulmonary inflammation in mice. Together, these findings provide the molecular basis for the specificity of SEFIR-SEFIR versus TIR-TIR domain interactions and consequent signaling. Moreover, we suggest that the CC' loop motif of SEFIR domains is a promising target for therapeutic strategies against inflammatory diseases associated with IL-17 or IL-25 signaling.

T cell-derived Act1 is necessary for IL-25-mediated Th2 responses and allergic airway inflammation.

The cellular and molecular mechanisms driven by IL-25 and its cognate receptor IL-17RB necessary for the promotion of Th2-mediating pathogenic pulmonary inflammation remains to be defined. We have previously reported the critical role of the U-box-type E3 ubiquitin ligase Act1 (1) for the downstream signaling of the IL-17 cytokine family including the Th2-promoting cytokine IL-25 (IL-17E) (2). In this study, we report that IL-25-driven but not conventional IL-4-driven Th2 polarization and cytokine production is impaired in Act1-deficient T cells. Also, Act1 deficiency in the T cell compartment results in the abrogation of eosinophilic airway infiltration as well as airway hyperresponsiveness in mouse models of Ag-induced airway inflammation. The in vivo generation of Ag-specific Th2 cytokine-producing cells is defective in the absence of Act1 expression in T cells after OVA/aluminum hydroxide immunization. Notably, the production of OVA-specific IgG(1) but not IgG(2a) or IgE is also impaired. At the molecular level, we report that IL-25-mediated induction of Th2 master regulator GATA-3 and the transcription factor GFI-1 is attenuated in Act1-deficient T cells. Taken together, our findings indicate that Act1 expression in T cells is required for cellular and humoral Th2-mediated allergic responses and the development of airway hyperresponsiveness, in part, through Act1's function in IL-25-induced development of Th2 T cells.

The inducible kinase IKKi is required for IL-17-dependent signaling associated with neutrophilia and pulmonary inflammation.

Interleukin 17 (IL-17) is critical in the pathogenesis of inflammatory and autoimmune diseases. Here we report that Act1, the key adaptor for the IL-17 receptor (IL-7R), formed a complex with the inducible kinase IKKi after stimulation with IL-17. Through the use of IKKi-deficient mice, we found that IKKi was required for IL-17-induced expression of genes encoding inflammatory molecules in primary airway epithelial cells, neutrophilia and pulmonary inflammation. IKKi deficiency abolished IL-17-induced formation of the complex of Act1 and the adaptors TRAF2 and TRAF5, activation of mitogen-activated protein kinases (MAPKs) and mRNA stability, whereas the Act1-TRAF6-transcription factor NF-κB axis was retained. IKKi was required for IL-17-induced phosphorylation of Act1 on Ser311, adjacent to a putative TRAF-binding motif. Substitution of the serine at position 311 with alanine impaired the IL-17-mediated Act1-TRAF2-TRAF5 interaction and gene expression. Thus, IKKi is a kinase newly identified as modulating IL-17 signaling through its effect on Act1 phosphorylation and consequent function.

Hyaluronan deposition and correlation with inflammation in a murine ovalbumin model of asthma.

Asthma is a chronic inflammatory disease of the airways characterized by airway remodeling, which includes changes in the extracellular matrix (ECM). However the role of the ECM in mediating these changes is poorly understood. Hyaluronan (HA), a major component of the ECM, has been implicated in asthma as well as in many other biological processes. Our study investigates the processes involved in HA synthesis, deposition, localization and degradation during an acute and chronic murine model of ovalbumin (OVA)-induced allergic pulmonary inflammation. Mice were sensitized, challenged to OVA and sacrificed at various time points during an 8-week challenge protocol. Bronchoalveolar lavage (BAL) fluids, blood, and lung tissue were collected for study. RNA, HA, protein and histopathology were analyzed. Analyses of lung sections and BAL fluids revealed an early deposition and an increase in HA levels within 24 h of antigen exposure. HA levels peaked at day 8 in BAL, while inflammatory cell recovery peaked at day 6. Hyaluronan synthase (HAS)1 and HAS2 on RNA levels peaked within 2 h of antigen exposure, while hyaluronidase (HYAL)1 and HYAL2 on RNA levels decreased. Both inflammatory cell infiltrates and collagen deposition co-localized with HA deposition within the lungs. These data support a role for HA in the pathogenesis of inflammation and airway remodeling in a murine model of asthma. HA deposition appears largely due to up regulation of HAS1 and HAS2. In addition, HA appears to provide the scaffolding for inflammatory cell accumulation as well as for new collagen synthesis and deposition.

Direct and differential suppression of myeloid-derived suppressor cell subsets by sunitinib is compartmentally constrained.

The antiangiogenic drug sunitinib is a receptor tyrosine kinase inhibitor with significant, yet not curative, therapeutic effects in metastatic renal cell carcinoma (RCC). Sunitinib is also an immunomodulator, potently reversing myeloid-derived suppressor cell (MDSC) accumulation and T-cell inhibition in the blood even of nonresponder RCC patients. We observed that sunitinib similarly prevented MDSC accumulation and restored normal T-cell function to the spleens of tumor-bearing mice, independent of the capacity of sunitinib to inhibit tumor progression (RENCA>CT26>4T1). Both monocytic and neutrophilic splenic MDSC were highly repressible by sunitinib. In contrast, MDSC within the microenvironment of 4T1 tumors or human RCC tumors proved highly resistant to sunitinib and ambient T-cell function remained suppressed. Proteomic analyses comparing tumor to peripheral compartments showed that granulocyte macrophage colony-stimulating factor (GM-CSF) predicted sunitinib resistance and recombinant GM-CSF conferred sunitinib resistance to MDSC in vivo and in vitro. MDSC conditioning with GM-CSF uniquely inhibited signal transducers and activators of transcription (STAT3) and promoted STAT5 activation. STAT5ab(null/null) MDSC were rendered sensitive to sunitinib in the presence of GM-CSF in vitro. We conclude that compartment-dependent GM-CSF exposure in resistant tumors may account for the regionalized effect of sunitinib upon host MDSC modulation and hypothesize that ancillary strategies to decrease such regionalized escape will enhance the potency of sunitinib as an immunomodulator and a cancer therapy.

Epithelium: the interplay between innate and Th2 immunity.

Mucosal epithelium functions not only as a physical barrier, but also as a regulator of innate and adaptive immune responses against foreign substances and microorganisms. In particular, epithelial cells have been directly implicated in Th2 responses, serving as a critical interface between innate immune responses and Th2 immunity. Emerging studies have revealed the cellular and molecular mechanisms by which the epithelium modulates Th2 responses through the production of a group of epithelial-derived Th2-driving cytokines, including interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin. These epithelial-derived Th2-driving cytokines execute a regulatory function of the epithelium on mucosal immunity by promoting Th2 responses and maintaining the balance of host immune homeostasis and defense against various pathogens. Dysregulation of these Th2-driving cytokines can lead to detrimental Th2-dependent inflammatory responses, often manifested in various forms of allergic and inflammatory diseases.