Search

Supplementary Figures and Legends

Clinical response rates after adoptive cell therapy (ACT) are highly correlated with in vivo persistence of the infused T cells. However, antigen-specific T cells found in tumor sites are often well-differentiated effector cells with limited persistence. Central memory CD8+ T cells, capable of self-renewal, represent desirable ACT products. We report here that exposure to a histone deacetylase inhibitor (HDACi) and IL21 could reprogram differentiated human CD8+ T cells into central memory–like T cells. Dedifferentiation of CD8+ T cells was initiated by increased H3 acetylation and chromatin accessibility at the CD28 promoter region. This led to IL21-mediated pSTAT3 binding to the CD28 region, and subsequent upregulation of surface CD28 and CD62L (markers of central memory T cells). The reprogrammed cells exhibited enhanced proliferation in response to both IL2 and IL15, and a stable memory-associated transcriptional signature (increased Lef1 and Tcf7). Our findings support the application of IL21 and HDACi for the in vitro generation of highly persistent T-cell populations that can augment the efficacy of adoptively transferred T cells.

PTEN loss in melanoma promotes resistance to T cell-mediated anti-tumor activity in the context of concurrent a selective inhibitor for mutant BRAF.

Supplementary Table S1. Patient characteristics of the single-agent anti-PD-1 cohort. Supplementary Table S2. List of primers used for gene expression analysis by real-time PCR. Supplementary Table S3. List of autophagy-related genes for overexpressing experiment.

PDF file - 30K, VEGF production by A375/H-2Db/gp100 melanoma cells

PDF file - 72K, PLX4720 treatment does not increase infiltration of adoptively transferred T cells into tumors containing WT BRAF

PDF file - 38K, mRNA levels of VEGF in patients with metastatic melanoma undergoing treatment with a selective inhibitor of BRAF(V600E)

PDF file - 82K, Administration of PLX4720 in vivo increases tumor infiltration of adoptively transferred T cells and enhances antitumor responses

PDF file - 24K, Pmel-1 T cells in the peripheral blood of tumor-bearing mice

PDF file - 26K, Chromium release assay

PDF file - 491K, Administration of PLX4720 in vivo increases tumor infiltration of adoptively transferred T cells

Purpose: Treatment of melanoma patients with selective BRAF inhibitors results in objective clinical responses in the majority of patients with BRAF-mutant tumors. However, resistance to these inhibitors develops within a few months. In this study, we test the hypothesis that BRAF inhibition in combination with adoptive T-cell transfer (ACT) will be more effective at inducing long-term clinical regressions of BRAF-mutant tumors.Experimental Design:BRAF-mutated human melanoma tumor cell lines transduced to express gp100 and H-2Db to allow recognition by gp100-specific pmel-1 T cells were used as xenograft models to assess melanocyte differentiation antigen–independent enhancement of immune responses by BRAF inhibitor PLX4720. Luciferase-expressing pmel-1 T cells were generated to monitor T-cell migration in vivo. The expression of VEGF was determined by ELISA, protein array, and immunohistochemistry. Importantly, VEGF expression after BRAF inhibition was tested in a set of patient samples.Results: We found that administration of PLX4720 significantly increased tumor infiltration of adoptively transferred T cells in vivo and enhanced the antitumor activity of ACT. This increased T-cell infiltration was primarily mediated by the ability of PLX4720 to inhibit melanoma tumor cell production of VEGF by reducing the binding of c-myc to the VEGF promoter. Furthermore, analysis of human melanoma patient tumor biopsies before and during BRAF inhibitor treatment showed downregulation of VEGF consistent with the preclinical murine model.Conclusion: These findings provide a strong rationale to evaluate the potential clinical application of combining BRAF inhibition with T-cell–based immunotherapy for the treatment of patients with melanoma. Clin Cancer Res; 19(2); 393–403. ©2012 AACR.

PDF file - 52K, PLX4720 exposure reduces binding of c-myc to the VEGF promoter in BRAF mutant melanoma cells

PDF file - 95K

PDF file - 95K, Tumor antigen expression is important for PLX4720-induced T cell infiltration into tumors

Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)–mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4–mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs.

Clinical response rates after adoptive cell therapy (ACT) are highly correlated with in vivo persistence of the infused T cells. However, antigen-specific T cells found in tumor sites are often well-differentiated effector cells with limited persistence. Central memory CD8+ T cells, capable of self-renewal, represent desirable ACT products. We report here that exposure to a histone deacetylase inhibitor (HDACi) and IL21 could reprogram differentiated human CD8+ T cells into central memory–like T cells. Dedifferentiation of CD8+ T cells was initiated by increased H3 acetylation and chromatin accessibility at the CD28 promoter region. This led to IL21-mediated pSTAT3 binding to the CD28 region, and subsequent upregulation of surface CD28 and CD62L (markers of central memory T cells). The reprogrammed cells exhibited enhanced proliferation in response to both IL2 and IL15, and a stable memory-associated transcriptional signature (increased Lef1 and Tcf7). Our findings support the application of IL21 and HDACi for the in vitro generation of highly persistent T-cell populations that can augment the efficacy of adoptively transferred T cells.

Background: Chronic inflammation, a hallmark of cancer, is associated with poor prognosis in human various malignancies. Predominantly, tumor associated neutrophils (TANs) and myeloid-derived suppressor cells (MDSCs) are immune cells well characterized to promote inflammation, support tumor growth and metastasis, by secreting chemokines, serine proteases and reactive oxygen species. Increased TANs and MDSCs are predictive markers of both tumor progression and metastasis, suggesting that their inhibition may provide viable anti-tumor strategies. Neutrophil elastase (NE), a serine protease exclusively secreted by TANs and MDSCs, is a crucial mediator of inflammation and tumor progression. Recent preliminary studies from our group suggests that genetic deletion of Elane (encoding NE) inhibits lung metastasis in in vivo models of breast cancer. Yet the precise mechanism(s) by which NE promotes tumorigenesis and metastasis of breast cancer remains to be elucidated. To address this gap in knowledge, the current focus of our work is to identify the tumor-intrinsic and -extrinsic mechanisms by which NE promotes metastasis. Methods: To determine the prognostic significance of NE, we analyzed 192 breast cancer patients (58% ER/PR+ve, 20%-HER-2 +ve and 22% TNBC) for infiltration of TANs. To examine the role of NE in breast cancer metastasis, we have generated Elane+/+ and Elane-/- mice in BALB/c and FVB/N genetic backgrounds, bearing 4T1 and PyMT tumors (orthotopic and spontaneous) respectively. Each of these mouse models develop lung metastasis in 80-90% in tumor-bearing mice within 1-3 months of primary tumor initiation. Results: Immunohistochemical staining of NE, a marker of TANs shows that higher infiltration of NE-positive TANs is associated with recurrence free survival with a hazard ratio of 3.4 (95% CI, 1.1-5.5). Genetic ablation of NE (Elane −/−) in the PyMT and 4T1 models strongly inhibits lung metastasis. Specifically, the number of lung foci was reduced by ~90% in PyMT Elane−/− mice compared to controls (21.8 vs. 2.7, respectively; p=0.0044). Similarly, ~50% decrease in the number of lung foci was observed in the 4T1 Elane−/− mice compared to control (7.8 vs. 3.4, respectively; p=0.0281). Comparison of inflammatory factors in the tumor microenvironment revealed that Csf3 (G-CSF), Cxcr2 (CXCR2) and Cxcl1 (CXCL1/KC) were significantly elevated in tumors from PyMT/4T1 Elane+/+ mice vs. PyMT/4T1 Elane-/- animals. Mechanistically, our result implies NE-dependent recruitment of immunosuppressive subsets (CXCL1 and related chemokines), thus sustaining inflammation at the primary tumor site. NE- dependent secretion of CXCL1 can further enrich for cancer stem-like cells (CSCs) via CXCR2 signaling, thus aiding metastasis. Lastly, we interrogated the efficacy reversible NE inhibitor AZD9668 in Elane+/+ mouse models. 100mg/kg daily treatment of AZD9668 reduced lung metastasis in PyMT mice by 94% compared to vehicle-treated mice (0.49+/- 0.21% vs. 0.03+/-0.01%; p=0.05). Conclusions: Collectively, our studies suggest that genetic and pharmacological ablation of NE reduces metastasis in in vivo models of breast cancer. NE inhibitors thus far have only been clinically tested in COPD patients and their efficacy in breast cancer remains to be evaluated. Our preclinical studies presented here are likely to provide the much needed rationale for the use of this class of NE inhibitors as a viable treatment strategy for the metastatic breast cancer. Citation Format: Amriti Rajender Lulla, Said Akli, Taylor T Chrisikos, Marc A. Pina, Yifan Zhou, Haiyan Li, Stephanie S Watowich, Khandan Keyomarsi. Neutrophil elastase as a therapeutic target to inhibit metastasis in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-02.

NF-κB, a family of transcription factors regulating diverse biological processes including immune responses, is activated by canonical and noncanonical pathways based on degradation of IκBα and processing of the IκB-like protein p100, respectively. Although p100 responds to noncanonical NF-κB stimuli for processing, it does not undergo degradation, but rather becomes accumulated, along with canonical NF-κB activation. We show here that the stability of p100 is tightly controlled by a deubiquitinase, Otub1. Otub1 deficiency not only promotes signal-induced p100 processing and noncanonical NF-κB activation but also causes steady-state p100 degradation, leading to aberrant NF-κB activation in the canonical pathway. B-cell-conditional deletion of Otub1 results in B-cell hyperplasia, antibody hyper-production, and lupus-like autoimmunity. Otub1-deficient B cells display aberrantly activated phenotypes and overproduce the cytokine IL-6, contributing to autoimmunity induction. Thus, maintenance of p100 stability by Otub1 serves as an unusual mechanism of NF-κB regulation that prevents autoimmunity.

CXCR5 mediates homing of both B and follicular helper T (TFH) cells into follicles of secondary lymphoid organs. We found that CXCR5+CD8+ T cells are present in human tonsils and follicular lymphoma, inhibit TFH-mediated B cell differentiation, and exhibit strong cytotoxic activity. Consistent with these findings, adoptive transfer of CXCR5+CD8+ T cells into an animal model of lymphoma resulted in significantly greater antitumor activity than CXCR5-CD8+ T cells. Furthermore, RNA-Seq-based transcriptional profiling revealed 77 differentially expressed genes unique to CXCR5+CD8+ T cells. Among these, a signature comprised of 33 upregulated genes correlated with improved survival in follicular lymphoma patients. We also showed that CXCR5+CD8+ T cells could be induced and expanded ex vivo using IL-23 plus TGF-β, suggesting a possible strategy to generate these cells for clinical application. In summary, our study identified CXCR5+CD8+ T cells as a distinct T cell subset with ability to suppress TFH-mediated B cell differentiation, exert strong antitumor activity, and confer favorable prognosis in follicular lymphoma patients.

Immunotherapies such as anti-CTLA-4 immune checkpoint blockade (ICB) have revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by off-target tissue damage or immune-related adverse events (irAEs). At present, there is limited understanding of irAE mechanisms, hampering development of approaches to mitigate their damage. We addressed this problem by generating animal models of intestinal irAE. Our results show that disruption of homeostatic immunity by genetic predisposition to intestinal inflammation or acute gastrointestinal infection sensitizes mice to anti-CTLA-4-mediated intestinal toxicity. Inflammation-prone mice treated with anti-CTLA-4 showed neutrophil accumulation, systemic interleukin-6 (IL-6) release, and dysbiosis. Significantly, IL-6 blockade combined with antibiotic treatment improved anti-CTLA-4 therapeutic efficacy and reduced intestinal irAEs. Immune signatures were validated in biopsies from patients who developed colitis during ICB, supporting the utility of our models. This study provides new pre-clinical models, mechanistic insight into irAEs, and potential approaches to enhance ICB efficacy while mitigating irAEs. Citation Format: Yifan Zhou, Yusra B. Medik, Bhakti Patel, Daniel B. Zamler, Sijie Chen, Thomas Chapman, Sarah Schneider, Rachel L. Babcock, Taylor T. Chrisikos, Laura M. Kahn, Allison M. Dyevoich, Elizabeth M. Park, Alexandria P. Cogdill, Daniel H. Johnson, Sarah B. Johnson, Khalida M. Wani, Debora A. Ledesma, Courtney W. Hudgens, Jingjing Wang, Md Abdul Wadud Khan, Aron Y. Joon, Weiyi Peng, Haiyan S. Li, Reetakshi Arora, Ximing Tang, Maria Gabriela Raso, Xuegong Zhang, Wai Chin Foo, Michael T. Tetzlaff, Gretchen E. Diehl, Karen Clise-Dwyer, Elizabeth M. Whitley, Matthew M. Gubin, James P. Allison, Patrick Hwu, Nadim J. Ajami, Adi Diab, Jennifer A. Wargo, Stephanie S. Watowich. Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5545.

Maintenance of immune homeostasis involves a synergistic relationship between the host and the microbiome. Canonical interferon (IFN) signaling controls responses to acute microbial infection, through engagement of the STAT1 transcription factor. However, the contribution of tonic levels of IFN to immune homeostasis in absence of acute infection remains largely unexplored. We report that STAT1 KO mice spontaneously developed an inflammatory disease marked by myeloid hyperplasia and splenic accumulation of hematopoietic stem cells. Moreover, these animals developed inflammatory bowel disease. Profiling gut bacteria revealed a profound dysbiosis in absence of tonic IFN signaling, which triggered expansion of TH17 cells and loss of splenic Treg cells. Resolution of dysbiosis by antibiotic treatment averted the TH17 bias, and blocking IL17 signaling prevented myeloid expansion and splenic stem cell accumulation. Thus, tonic IFNs regulate gut microbial ecology, which is crucial for maintaining physiologic immune homeostasis and preventing inflammation.

Background Type 1 conventional dendritic cells (cDC1s) possess efficient antigen presentation and cross-presentation activity, as well as potent T cell priming ability. Tissue-resident cDC1s (CD103+ cDC1s in mice, CD141+ cDC1s in humans) are linked with improved tumor control, yet the efficacy of immunotherapy using this population is understudied. Methods We generated murine CD103+ cDC1s in vitro and examined their expression of cDC1-related factors, antigen cross-presentation activity, and accumulation in tumor-draining lymph nodes (TdLNs). The antitumor efficacy of the in vitro-generated CD103+ cDC1s was studied in murine melanoma and osteosarcoma models. We evaluated tumor responses on vaccination with CD103+ cDC1s, compared these to vaccination with monocyte-derived DCs (MoDCs), tested CD103+ cDC1 vaccination with checkpoint blockade, and examined the antimetastatic activity of CD103+ cDC1s. Results In vitro-generated CD103+ cDC1s produced cDC1-associated factors such as interleukin-12p70 and CXCL10, and demonstrated antigen cross-presentation activity on stimulation with the toll-like receptor 3 agonist polyinosinic:polycytidylic acid (poly I:C). In vitro-generated CD103+ cDC1s also migrated to TdLNs following poly I:C treatment and intratumoral delivery. Vaccination with poly I:C-activated and tumor antigen-loaded CD103+ cDC1s enhanced tumor infiltration of tumor antigen-specific and interferon-γ+ CD8+ T cells, and suppressed melanoma and osteosarcoma growth. CD103+ cDC1s showed superior antitumor efficacy compared with MoDC vaccination, and led to complete regression of 100% of osteosarcoma tumors in combination with CTLA-4 antibody-mediated checkpoint blockade. In vitro-generated CD103+ cDC1s effectively protected mice from pulmonary melanoma and osteosarcoma metastases. Conclusions Our data indicate an in vitro-generated CD103+ cDC1 vaccine elicits systemic and long-lasting tumor-specific T cell-mediated cytotoxicity, which restrains primary and metastatic tumor growth. The CD103+ cDC1 vaccine was superior to MoDCs and enhanced response to immune checkpoint blockade. These results indicate the potential for new immunotherapies based on use of cDC1s alone or in combination with checkpoint blockade.

Xiao et al. demonstrate that a protein kinase, TBK1, regulates the function of dendritic cells in mediating immune responses., Dendritic cells (DCs) are crucial for mediating immune responses but, when deregulated, also contribute to immunological disorders, such as autoimmunity. The molecular mechanism underlying the function of DCs is incompletely understood. In this study, we have identified TANK-binding kinase 1 (TBK1), a master innate immune kinase, as an important regulator of DC function. DC-specific deletion of Tbk1 causes T cell activation and autoimmune symptoms and also enhances antitumor immunity in animal models of cancer immunotherapy. The TBK1-deficient DCs have up-regulated expression of co-stimulatory molecules and increased T cell–priming activity. We further demonstrate that TBK1 negatively regulates the induction of a subset of genes by type I interferon receptor (IFNAR). Deletion of IFNAR1 could largely prevent aberrant T cell activation and autoimmunity in DC-conditional Tbk1 knockout mice. These findings identify a DC-specific function of TBK1 in the maintenance of immune homeostasis and tolerance.

Conventional dendritic cells (cDCs) are a critical immune population, composed of multiple subsets, and responsible for controlling adaptive immunity and tolerance. Although migratory type 1 cDCs (CD103+ cDC1s in mice) are necessary to mount CD8+ T cell-mediated anti-tumor immunity, whether and how tumors modulate CD103+ cDC1 function remain understudied. Signal Transducer and Activator of Transcription 3 (STAT3) mediates the intracellular signaling of tumor-associated immunosuppressive cytokines, such as interleukin (IL)-10; thus, we hypothesized that STAT3 restrained anti-tumor immune responses elicited by CD103+ cDC1s. Herein, we show that in vitro-derived STAT3-deficient (Stat3∆/∆) CD103+ cDC1s are refractory to the inhibitory effects of IL-10 on Toll-like receptor 3 (TLR3) agonist-induced maturation responses. In a tumor vaccination approach, we found Stat3∆/∆ CD103+ cDC1s restrained mammary gland tumor growth and increased mouse survival more effectively than STAT3-sufficient CD103+ cDC1s. In addition, vaccination with Stat3∆/∆ CD103+ cDC1s elicited increased amounts of tumor antigen-specific CD8+ T cells and IFN-γ+ CD4+ T cells in tumors and tumor-draining lymph nodes versus phosphate-buffered saline (PBS)-treated animals. Furthermore, IL-10 receptor-deficient CD103+ cDC1s controlled tumor growth to a similar degree as Stat3∆/∆ CD103+ cDC1s. Taken together, our data reveal an inhibitory role for STAT3 in CD103+ cDC1 maturation and regulation of anti-tumor immunity. Our results also suggest IL-10 is a key factor eliciting immunosuppressive STAT3 signaling in CD103+ cDC1s in breast cancer. Thus, inhibition of STAT3 in cDC1s may provide an important strategy to improve their efficacy in tumor vaccination approaches and cDC1-mediated control of anti-tumor immunity.

Clinical response rates after adoptive cell therapy (ACT) are highly correlated with in vivo persistence of the infused T cells. However, antigen-specific T cells found in tumor sites are often well-differentiated effector cells with limited persistence. Central memory CD8(+) T cells, capable of self-renewal, represent desirable ACT products. We report here that exposure to a histone deacetylase inhibitor (HDACi) and IL21 could reprogram differentiated human CD8(+) T cells into central memory-like T cells. De-differentiation of CD8(+) T cells was initiated by increased H3 acetylation and chromatin accessibility at the CD28 promoter region. This led to IL21-mediated pSTAT3 binding to the CD28 region, and subsequent upregulation of surface CD28 and CD62L (markers of central memory T cells). The reprogrammed cells exhibited enhanced proliferation in response to both IL2 and IL15, and a stable memory-associated transcriptional signature (increased Lef1 and Tcf7). Our findings support the application of IL21 and HDACi for the in vitro generation of highly persistent T cell populations that can augment the efficacy of adoptively transferred T cells.

Type 1 conventional dendritic cells (cDC1s) have critical roles in inducing adaptive immune responses and mediating immune tolerance. Signal Transducer and Activator of Transcription 5 (STAT5) was found to be crucial for monocyte-derived DC development; however, its role in non-lymphoid tissue CD103+cDC1s remained largely unknown. We evaluated the role of STAT5 by employing myeloid-specific Stat5-deficient mice,LysM-Cre +Stat5f/f and CD11c-Cre+Stat5f/f mice. Both strains show deficiencies in the numbers of CD103+cDC1s and alveolar macrophages (AMs) in lung, suggesting ineffective maintenance of lung homeostasis. Consistent with this concept, CD11c-Cre+Stat5f/f mice show granulocyte and monocyte accumulation in lung, accompanied by a reduction in the number of CD4+T, CD8+T, and B cells. Moreover, CD11c-Cre+Stat5-deficient mice have increased numbers of alveolar histiocytes, which are also enlarged, as well as accumulation of amorphous and eosinophilic material in alveolar spaces, alveolar histiocytosis, and proteinosis. In addition to the lung, liver CD103+cDC1s were also decreased by Stat5 depletion, however colon CD103+cDC1s were not affected. To examine cell-autonomous roles for STAT5, we generated Stat5-deficient CD103+cDC1s using our established bone marrow culture system, and tested in vitro-differentiated CD103+cDC1s for antigen presentation activity. These assays suggest Stat5 deficiency impaired CD103+cDC1s antigen cross-presentation ability. Taken together, our study showed that STAT5 is required for homeostasis and function of CD103+cDC1s in non-lymphoid organs.

T cell–mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. We show that loss of PTEN in tumor cells in preclinical models of melanoma inhibits T cell–mediated tumor killing and decreases T-cell trafficking into tumors. In patients, PTEN loss correlates with decreased T-cell infiltration at tumor sites, reduced likelihood of successful T-cell expansion from resected tumors, and inferior outcomes with PD-1 inhibitor therapy. PTEN loss in tumor cells increased the expression of immunosuppressive cytokines, resulting in decreased T-cell infiltration in tumors, and inhibited autophagy, which decreased T cell–mediated cell death. Treatment with a selective PI3Kβ inhibitor improved the efficacy of both anti–PD-1 and anti–CTLA-4 antibodies in murine models. Together, these findings demonstrate that PTEN loss promotes immune resistance and support the rationale to explore combinations of immunotherapies and PI3K–AKT pathway inhibitors. Significance: This study adds to the growing evidence that oncogenic pathways in tumors can promote resistance to the antitumor immune response. As PTEN loss and PI3K–AKT pathway activation occur in multiple tumor types, the results support the rationale to further evaluate combinatorial strategies targeting the PI3K–AKT pathway to increase the efficacy of immunotherapy. Cancer Discov; 6(2); 202–16. ©2015 AACR. See related commentary by Rizvi and Chan, p. 128. This article is highlighted in the In This Issue feature, p. 109

Heterozygous mutations in the transcriptional regulator GATA-2 associate with multilineage immunodeficiency, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML). The majority of these mutations localize in the zinc finger (ZnF) domains, which mediate GATA-2 DNA binding. Deregulated hematopoiesis with GATA-2 mutation frequently develops in adulthood, yet GATA-2 function in the bone marrow remains unresolved. To investigate this, we conditionally deleted the GATA-2 C-terminal ZnF (C-ZnF) coding sequences in adult mice. Upon Gata2 C-ZnF deletion, we observed rapid peripheral cytopenia, bone marrow failure, and decreased c-Kit expression on hematopoietic progenitors. Transplant studies indicated GATA-2 has a cell-autonomous role in bone marrow hematopoiesis. Moreover, myeloid lineage populations were particularly sensitive to Gata2 hemizygosity, while molecular assays indicated GATA-2 regulates c-Kit expression in multilineage progenitor cells. Enforced c-Kit expression in Gata2 C-ZnF-deficient hematopoietic progenitors enhanced myeloid colony activity, suggesting GATA-2 sustains myelopoiesis via a cell intrinsic role involving maintenance of c-Kit expression. Our results provide insight into mechanisms regulating hematopoiesis in bone marrow and may contribute to a better understanding of immunodeficiency and bone marrow failure associated with GATA-2 mutation.

Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-I) producing cells that mediate anti-viral responses, anti-tumor immunity, and autoimmunity. When exposed to Toll-like receptor 7 (TLR7)- and TLR9-ligands, pDCs mature and produce IFN-Is; pDCs also acquire conventional DC (cDC)-like morphology and features, including the cell surface expression of antigen presentation and co-stimulatory molecules, secretion of additional cytokines, and ability to activate adaptive T cell responses. Yet, the transcriptional pathways regulating TLR-induced pDC maturation are poorly characterized. We found the mRNA and protein expression of the inhibitor of DNA binding protein 2 (Id2) was induced in TLR7/9-stimulated pDCs, and Id2 expression correlated with greater cDC-like features. Id2 normally blocks pDC development by antagonizing the pDC-master regulator, E2-2, while E2-2 suppresses Id2 transcription. Thus, we hypothesize TLR-activated signaling pathways in pDCs control the abundance of transcriptional regulators (e.g., E2-2) at the Id2 promoter as well as the promoter chromatin state, leading to induction of Id2 transcription during pDC maturation. Using genetic knockout mouse models, we found Id2 induction was independent of interferon alpha receptor and nuclear factor kappa beta signaling pathways. Chromatin-immunoprecipitation assays suggested E2-2 abundance at the Id2 promoter decreased following TLR stimulation, while activating histone modifications increased. These data are consistent with transcriptional induction of Id2 upon TLR stimulation. Future work will investigate the effect of additional regulators and TLR-regulated pathways on Id2 induction in TLR7/9-stimulated pDCs.

Type 1 conventional dendritic cells (cDC1s) possess efficient antigen presentation and cross-presentation activity, yet the efficacy of immunotherapy utilizingthis population is understudied. We used in vitro-generated CD103+cDC1s in vaccination strategies to test their ability to control melanoma and osteosarcoma tumors. In vitro-generated CD103+cDC1s produced cDC1-associated factors such as IL-12p70 and CXCL10, and demonstrated antigen cross-presentation activity upon stimulation with the Toll-like receptor 3 (TLR3) agonist polyinosinic:polycytidylic acid (poly I:C). In vitro-generated CD103+cDC1s also migrated to tumor-draining lymph nodes (TdLNs) following poly I:C treatment and intratumoral (i.t.) delivery. Vaccination with poly I:C-activated and tumor antigen-loaded CD103+cDC1s enhanced tumor infiltration of tumor antigen-specific and IFN-g+CD8+ T cells, and suppressed melanoma and osteosarcoma growth. CD103+cDC1s showed superior anti-tumor efficacy compared to vaccination with monocyte-derived DCs (MoDCs), and led to complete regression of 100% of osteosarcoma tumors in combination with CTLA-4 antibody-mediated checkpoint blockade. In addition, CD103+cDC1s effectively protected mice from pulmonary melanoma and osteosarcoma metastases. Our data indicate an in vitro-generated CD103+cDC1 vaccine elicits systemic and long-lasting tumor-specific T cell-mediated cytotoxicity, which restrains primary and metastatic tumor growth. The CD103+cDC1 vaccine was superior to MoDCs and enhanced response to immune checkpoint blockade. These results indicate the potential for new immunotherapies based on use of cDC1s alone or in combination with checkpoint blockade.

Intestinal infections trigger immune responses that in some cases become dysregulated and lead to acute or chronic disease; understanding immune regulatory mechanisms in infection will help advance new therapies for intestinal disorders. Citrobacter rodentium infection in mice mimics enteropathogenic Escherichia coli infections in humans. We found that Leukemia inhibitory factor (LIF), a member of the interleukin-6 (IL-6) cytokine family, is upregulated in colon and serum following C. rodentium infection; however, the source, targets and effects of this cytokine during intestinal disease remain unclear. To investigate the role of LIF during infection, we generated mice with LIF receptor (LIFR) deficiency in endothelial and hematopoietic cells (Tie2cre Lifrf/f) or CD11c+ cells (CD11c cre Lifrf/f). Upon C. rodentium infection, Tie2cre Lifrf/fandCD11c cre Lifrf/f mice had reduced colon length, increased bacterial dissemination to spleen and liver, and greater colon pathology scores relative to infected controls. Comparison of cytokine and chemokine expression in the colon revealed upregulation of various factors in infected LIFR-deficient mice, with the pro-inflammatory cytokine IL-23 significantly increased in CD11c cre Lifrf/f mice. Our preliminary data from LIFR-deficient, bone marrow-derived myeloid cells suggests LIFR signaling inhibits inflammatory cytokine production in vitro. Collectively, our results suggest LIFR signaling in CD11c+ cells inhibits colonic IL-23 production and protects from immune dysregulation during C. rodentium infection. Our current studies are investigating LIF-producing and -responding populations in colon, to elucidate protective immune mechanisms during infection.

Conventional dendritic cells (cDCs) are a crucial immune population, which includes multiple subtypes that coordinate adaptive immunity and tolerance. Migratory type 1 cDC1s (CD103+ cDC1s in mice) are required to induce CD8+ T cell-mediated anti-tumor immunity, yet whether and how tumors regulate CD103+ cDC1 function is largely unknown. Many tumor-associated immunosuppressive cytokines, such as interleukin (IL)-10, require Signal Transducer and Activator of Transcription 3 (STAT3) for intracellular signaling; therefore, we sought to determine whether STAT3 inhibits CD103+ cDC1-mediated anti-tumor immunity. Indeed, we found that in vitro-derived STAT3-deficient (Stat3Δ/Δ) CD103+ cDC1sare resistant to IL-10 – mediated inhibition of Toll-like receptor 3 (TLR3)-agonist induced maturation. When used as an anti-tumor vaccine, Stat3Δ/Δ CD103+ cDC1s inhibited breast tumor growth and increased mouse survival to a greater degree than STAT3-sufficient CD103+ cDC1s. Vaccination with Stat3Δ/Δ CD103+ cDC1s increased tumor antigen-specific CD8+ T cells and IFN-g+ CD4+ T cells in tumors and tumor-draining lymph nodes compared to controls. Moreover, IL-10 receptor-deficient CD103+ cDC1s restrained tumor growth as effectively as Stat3Δ/Δ CD103+ cDC1s. Taken together, our results demonstrate STAT3 suppresses CD103+ cDC1 maturation and vaccine efficacy in breast cancer. The data also suggest IL-10 is an important factor in the murine mammary tumor environment mediating immunosuppressive STAT3 signaling in CD103+ cDC1s. Thus, STAT3 inhibition may provide an effective strategy for improving the anti-tumor efficacy of CD103+ cDC1-based vaccines.

BackgroundType 1 conventional dendritic cells (cDC1s) possess efficient antigen presentation and cross-presentation activity, as well as potent T cell priming ability. Tissue-resident cDC1s (CD103+cDC1s in mice, CD141+cDC1s in humans) are linked with improved tumor control, yet the efficacy of immunotherapy using this population is understudied.MethodsWe generated murine CD103+cDC1s in vitro and examined their expression of cDC1-related factors, antigen cross-presentation activity, and accumulation in tumor-draining lymph nodes (TdLNs). The antitumor efficacy of the in vitro-generated CD103+cDC1s was studied in murine melanoma and osteosarcoma models. We evaluated tumor responses on vaccination with CD103+cDC1s, compared these to vaccination with monocyte-derived DCs (MoDCs), tested CD103+cDC1 vaccination with checkpoint blockade, and examined the antimetastatic activity of CD103+cDC1s.ResultsIn vitro-generated CD103+cDC1s produced cDC1-associated factors such as interleukin-12p70 and CXCL10, and demonstrated antigen cross-presentation activity on stimulation with the toll-like receptor 3 agonist polyinosinic:polycytidylic acid (poly I:C). In vitro-generated CD103+cDC1s also migrated to TdLNs following poly I:C treatment and intratumoral delivery. Vaccination with poly I:C-activated and tumor antigen-loaded CD103+cDC1s enhanced tumor infiltration of tumor antigen-specific and interferon-γ+CD8+T cells, and suppressed melanoma and osteosarcoma growth. CD103+cDC1s showed superior antitumor efficacy compared with MoDC vaccination, and led to complete regression of 100% of osteosarcoma tumors in combination with CTLA-4 antibody-mediated checkpoint blockade. In vitro-generated CD103+cDC1s effectively protected mice from pulmonary melanoma and osteosarcoma metastases.ConclusionsOur data indicate an in vitro-generated CD103+cDC1 vaccine elicits systemic and long-lasting tumor-specific T cell-mediated cytotoxicity, which restrains primary and metastatic tumor growth. The CD103+cDC1 vaccine was superior to MoDCs and enhanced response to immune checkpoint blockade. These results indicate the potential for new immunotherapies based on use of cDC1s alone or in combination with checkpoint blockade.

Conventional dendritic cells (cDCs) are a critical immune population, composed of multiple subsets, and responsible for controlling adaptive immunity and tolerance. Although migratory type 1 cDCs (CD103+ cDC1s in mice) are necessary to mount CD8+ T cell-mediated anti-tumor immunity, whether and how tumors modulate CD103+ cDC1 function remain understudied. Signal Transducer and Activator of Transcription 3 (STAT3) mediates the intracellular signaling of tumor-associated immunosuppressive cytokines, such as interleukin (IL)-10, thus, we hypothesized that STAT3 restrained anti-tumor immune responses elicited by CD103+ cDC1s. Herein, we show that in vitro-derived STAT3-deficient (Stat3∆/∆) CD103+ cDC1s are refractory to the inhibitory effects of IL-10 on Toll-like receptor 3 (TLR3) agonist-induced maturation responses. In a tumor vaccination approach, we found Stat3∆/∆ CD103+ cDC1s restrained mammary gland tumor growth and increased mouse survival more effectively than STAT3-sufficient CD103+ cDC1s. In addition, vaccination with Stat3∆/∆ CD103+ cDC1s elicited increased amounts of tumor antigen-specific CD8+ T cells and IFN-&gamma, + CD4+ T cells in tumors and tumor-draining lymph nodes versus phosphate-buffered saline (PBS)-treated animals. Furthermore, IL-10 receptor-deficient CD103+ cDC1s controlled tumor growth to a similar degree as Stat3∆/∆ CD103+ cDC1s. Taken together, our data reveal an inhibitory role for STAT3 in CD103+ cDC1 maturation and regulation of anti-tumor immunity. Our results also suggest IL-10 is a key factor eliciting immunosuppressive STAT3 signaling in CD103+ cDC1s in breast cancer. Thus, inhibition of STAT3 in cDC1s may provide an important strategy to improve their efficacy in tumor vaccination approaches and cDC1-mediated control of anti-tumor immunity.

BACKGROUND & AIMS: The peroxisome proliferator activated receptor delta (PPARD) regulates cell metabolism, proliferation, and inflammation and has been associated with gastric and other cancers. Villin-positive epithelial cells are a small population of quiescent gastric progenitor cells. We expressed PPARD from a villin promoter to investigate the role of these cells and PPARD in development of gastric cancer. METHODS: We analyzed gastric tissues from mice that express the Ppard (PPARD1 and PPARD2 mice) from a villin promoter, and mice that did not carry this transgene (controls), by histology and immunohistochemistry. We performed cell lineage tracing experiments and analyzed the microbiomes, chemokine and cytokine production, and immune cells and transcriptomes of stomachs of these mice. We also performed immunohistochemical analysis of PPARD levels in in 2 sets of human gastric tissue microarrays. RESULTS: Thirty-eight percent of PPARD mice developed spontaneous, invasive gastric adenocarcinomas, with severe chronic inflammation. Levels of PPARD were increased in human gastric cancer tissues, compared with non-tumor tissues, and associated with gastric cancer stage and grade. We found an inverse correlation between level of PPARD in tumor tissue and patients survival time. Gastric microbiomes from PPARD and control mice did not differ significantly. Lineage-tracing experiments identified villin-expressing gastric progenitor cells (VGPCs) as the origin of gastric tumors in PPARD mice. In these mice, PPARD upregulated CCL20 and CXCL1, which increased infiltration of the gastric mucosa by immune cells. Immune cell production of inflammatory cytokines promoted chronic gastric inflammation and expansion and transformation of VGPCs, leading to tumorigenesis. We identified a positive-feedback loop between PPARD and interferon gamma signaling that sustained gastric inflammation to induce VGPC transformation and gastric carcinogenesis. CONCLUSIONS: We found PPARD overexpression in VPGCs to result in inflammation, dysplasia, and tumor formation. PPARD and VGPCs might be therapeutic targets for stomach cancer.

Blood cell formation must be appropriately maintained throughout life to provide robust immune function, hemostasis, and oxygen delivery to tissues, and to prevent disorders that result from over- or underproduction of critical lineages. Persistent inflammation deregulates hematopoiesis by damaging hematopoietic stem and progenitor cells (HSPCs), leading to elevated myeloid cell output and eventual bone marrow failure. Nonetheless, antiinflammatory mechanisms that protect the hematopoietic system are understudied. The transcriptional regulator STAT3 has myriad roles in HSPC-derived populations and nonhematopoietic tissues, including a potent antiinflammatory function in differentiated myeloid cells. STAT3 antiinflammatory activity is facilitated by STAT3-mediated transcriptional repression of Ube2n, which encodes the E2 ubiquitin-conjugating enzyme Ubc13 involved in proinflammatory signaling. Here we demonstrate a crucial role for STAT3 antiinflammatory activity in preservation of HSPCs and lineage-balanced hematopoiesis. Conditional Stat3 removal from the hematopoietic system led to depletion of the bone marrow lineage− Sca-1+ c-Kit+ CD150+ CD48− HSPC subset (LSK CD150+ CD48− cells), myeloid-skewed hematopoiesis, and accrual of DNA damage in HSPCs. These responses were accompanied by intrinsic transcriptional alterations in HSPCs, including deregulation of inflammatory, survival and developmental pathways. Concomitant Ube2n/Ubc13 deletion from Stat3-deficient hematopoietic cells enabled lineage-balanced hematopoiesis, mitigated depletion of bone marrow LSK CD150+ CD48− cells, alleviated HSPC DNA damage, and corrected a majority of aberrant transcriptional responses. These results indicate an intrinsic protective role for STAT3 in the hematopoietic system, and suggest that this is mediated by STAT3-dependent restraint of excessive proinflammatory signaling via Ubc13 modulation.

// Mingjun Zhang 1, 2 , Jin He 1 , Zhiqiang Liu 1 , Yong Lu 1, 2 , Yuhuan Zheng 1, 2 , Haiyan Li 1, 2 , Jingda Xu 1 , Huan Liu 1 , Jianfei Qian 1, 2 , Robert Z. Orlowski 1 , Larry W. Kwak 1 , Qing Yi 1, 2 , Jing Yang 1, 3 1 Department of Lymphoma/Myeloma, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 2 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA 3 Cancer Research Institute and Cancer Hospital, Guangzhou Medical University, Guangzhou, China Correspondence to: Jing Yang, e-mail: jiyang@mdanderson.org Qing Yi, e-mail: yiq@ccf.org Keywords: multiple myeloma, anti-β 2 M monoclonal antibody, bortezomib, autophagy, NF-κ p65 Received: November 17, 2014 Accepted: January 29, 2015 Published: March 31, 2015 ABSTRACT Our previous studies showed that anti-β 2 M monoclonal antibodies (mAbs) have strong and direct apoptotic effects on multiple myeloma (MM) cells, suggesting that anti-β 2 M mAbs might be developed as a novel therapeutic agent. In this study, we investigated the anti-MM effects of combination treatment with anti-β 2 M mAbs and bortezomib (BTZ). Our results showed that anti-β 2 M mAbs enhanced BTZ-induced apoptosis of MM cell lines and primary MM cells. Combination treatment could also induce apoptosis of BTZ-resistant MM cells, and the enhanced effect depended on the surface expression of β 2 M on MM cells. BTZ up-regulated the expression of autophagy proteins, whereas combination with anti-β 2 M mAbs inhibited autophagy. Sequence analysis of the promoter region of beclin 1 identified 3 putative NF-κB-binding sites from –615 to –789 bp. BTZ treatment increased, whereas combination with anti-β 2 M mAbs reduced, NF-κB transcription activities in MM cells, and combination treatment inhibited NF-κB p65 binding to the beclin 1 promoter. Furthermore, anti-β 2 M mAbs and BTZ combination treatment had anti-MM activities in an established MM mouse model. Thus, our studies provide new insight and support for the clinical development of an anti-β 2 M mAb and BTZ combination treatment to overcome BTZ drug resistance and improve MM patient survival.

Dendritic cells (DCs) are the principal antigen-presenting cells of the immune system and play key roles in controlling immune tolerance and activation. As such, DCs are chief mediators of tumor immunity. DCs can regulate tolerogenic immune responses that facilitate unchecked tumor growth. Importantly, however, DCs also mediate immune-stimulatory activity that restrains tumor progression. For instance, emerging evidence indicates the cDC1 subset has important functions in delivering tumor antigens to lymph nodes and inducing antigen-specific lymphocyte responses to tumors. Moreover, DCs control specific therapeutic responses in cancer including those resulting from immune checkpoint blockade. DC generation and function is influenced profoundly by cytokines, as well as their intracellular signaling proteins including STAT transcription factors. Regardless, our understanding of DC regulation in the cytokine-rich tumor microenvironment is still developing and must be better defined to advance cancer treatment. Here, we review literature focused on the molecular control of DCs, with a particular emphasis on cytokine- and STAT-mediated DC regulation. In addition, we highlight recent studies that delineate the importance of DCs in anti-tumor immunity and immune therapy, with the overall goal of improving knowledge of tumor-associated factors and intrinsic DC signaling cascades that influence DC function in cancer.

MicroRNA-22 (miR-22) is a highly conserved microRNA that can regulate cell proliferation, oncogenesis, and cell maturation, especially during stress. In hematopoietic stem cells (HSCs) miR-22 has been reported to be involved in the regulation of key self-renewal factors including Tet2. Recent work demonstrates that miR-22 also participates in regulation of the interferon response, and expression profiling studies suggest that it is variably expressed at different stages in erythroid differentiation. We thus hypothesized that miR-22 regulates maturation of erythroid progenitors during stress hematopoiesis through its interaction with interferon. We compared the blood and bone marrow of wild type (WT) and miR-22-deficient mice at baseline and upon infectious challenge with systemic lymphochoriomeningitis (LCMV) virus. MiR-22-deficient mice maintained platelet counts better than WT mice during infection, but they showed significantly reduced red blood cells (RBC) and hemoglobin. Analysis of bone marrow progenitors demonstrated better overall survival and improved HSC homeostasis in infected miR-22-null mice compared to WT, attributable to a blunted interferon response to LCMV challenge in the miR-22-null mice. We found that miR-22 was exclusively expressed in stage II erythroid precursors and was downregulated upon infection in WT mice. Our results indicate that miR-22 promotes the interferon response to viral infection and that it functions at baseline as a brake to slow erythroid differentiation and maintain adequate erythroid potential. Impaired regulation of erythrogenesis in the absence of miR-22 can lead to anemia during infection.

// Fei Mao 1,* , Qiang Tu 2,* , Li Wang 1 , Fuliang Chu 3 , Xia Li 4 , Haiyan S. Li 5 and Wenrong Xu 1 1 Key Laboratory of Medical Science and Laboratory Medicine of Jiangsu Province, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, P.R. China 2 Jiangning Hospital of Nanjing, Nanjing, Jiangsu, P.R. China 3 Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 4 Department of Gastroenterology, Binzhou Medical University Yantai Affiliated Hospital, Yantai, Shandong, P.R. China 5 Department of Immunology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA * These authors have contributed equally to this work Correspondence to: Wenrong Xu, email: // Haiyan S. Li, email: // Keywords : mesenchymal stem cell, inflammatory bowel disease, cell therapy, pericyte, tissue repair Received : January 05, 2017 Accepted : March 06, 2017 Published : March 29, 2017 Abstract Mesenchymal stem or stromal cells (MSCs) are non-hematopoietic stem cells that facilitate tissue regeneration through mechanisms involving self-renewal and differentiation, supporting angiogenesis and tissue cell survival, and limiting inflammation. MSCs were originally identified and expanded in long-term cultures of cells from bone marrow and other organs; and their native identity was recently confined into pericytes and adventitial cells in vascularized tissue. The multipotency, as well as the trophic and immunosuppressive effects, of MSCs have prompted the rapid development of clinical applications for many diseases involving tissue inflammation and immune disorders, including inflammatory bowel disease. Although standard criteria have been established to define MSCs, their therapeutic efficacy has varied significantly among studies due to their natural heterogenicity. Thus, understanding the biological and immunological features of MSCs is critical to standardize and optimize MSCs-based therapy. In this review, we highlight the cellular and molecular mechanisms involved in MSCs-mediated tissue repair and immunosuppression. We also provide an update on the current development of MSCs-based clinical trials, with a detailed discussion of MSC-based cell therapy in inflammatory bowel disease.

SummaryProduction of type I interferons (IFN-I) is a crucial innate immune mechanism against viral infections. IFN-I induction is subject to negative regulation by both viral and cellular factors, but the underlying mechanism remains unclear. We report that the noncanonical NF-κB pathway was stimulated along with innate immune cell differentiation and viral infections and had a vital role in negatively regulating IFN-I induction. Genetic deficiencies in major components of the noncanonical NF-κB pathway caused IFN-I hyperinduction and rendered cells and mice substantially more resistant to viral infection. Noncanonical NF-κB suppressed signal-induced histone modifications at the Ifnb promoter, an action that involved attenuated recruitment of the transcription factor RelA and a histone demethylase, JMJD2A. These findings reveal an unexpected function of the noncanonical NF-κB pathway and highlight an important mechanism regulating antiviral innate immunity.

Our previous studies showed that anti-β2M monoclonal antibodies (mAbs) at high doses have direct apoptotic effects on myeloma cells, suggesting that anti-β2M mAbs might be developed as a novel therapeutic agent. In this study, we investigated the ability of the mAbs at much lower concentrations to indirectly kill myeloma cells by utilizing immune effector cells or molecules. Our results showed that anti-β2M mAbs effectively lysed MM cells via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which were correlated with and dependent on the surface expression of β2M on MM cells. The presence of MM bone marrow stromal cells or addition of IL-6 did not attenuate anti-β2M mAb-induced ADCC and CDC activities against MM cells. Furthermore, anti-β2M mAbs only showed limited cytotoxicity toward normal B cells and nontumorous mesenchymal stem cells, indicating that the ADCC and CDC activities of the anti-β2M mAbs were more prone to the tumor cells. Lenalidomide potentiated in vitro ADCC activity against MM cells and in vivo tumor inhibition capacity induced by the anti-β2M mAbs by enhancing the activity of NK cells. These results support clinical development of anti-β2M mAbs, both as a monotherapy and in combination with lenalidomide, to improve MM patient outcome.

Significance Our laboratory has identified a critical role of IL-9 in promoting endogenous tumor-specific cytotoxic T lymphocyte response. In this study, we found that differentiation of CD8 + T cells under T helper 9-polarizing conditions induces the development of an IL-9–producing less cytolytic IL-9–skewed CD8 + T (Tc9) cell subset. Noticeably, adoptive transfer of tumor-reactive Tc9 cells elicited greater antitumor responses against large established tumors than classic type-I CD8 + cytotoxic T cells that are used in clinical protocols. Importantly, Tc9 cells have substantially enhanced persistence potential and possess the capacity to acquire/maintain effector function after transfer. Our results also revealed that Tc9-mediated therapeutic effect critically depended on IL-9 production in vivo. The ability of Tc9 cells to confer sustained antitumor responses might open an avenue for the advances of cancer immunotherapy.

G-CSF stimulates the expression of the chemokine MIP-2 in mature neutrophils by a direct transcriptional mechanism dependent upon STAT3. Neutrophil mobilization from the bone marrow is a critical aspect of the innate immune response, enabling a rapid deployment of phagocytes to infected or inflamed tissue. The cytokine G-CSF, which is induced rapidly during infection, elicits a swift and potent mobilizing response, yet its mechanisms of action remain poorly understood. Here, we studied the role of G-CSF and its principal signal transducer STAT3 in regulating expression of the neutrophil chemoattractant MIP-2. Our studies revealed Gr-1hi mature neutrophils as major sources of Cxcl2 (MIP-2) mRNA in bone marrow and G-CSF-responsive MIP-2 protein production. Induction of Cxcl2 was regulated directly by G-CSF-activated STAT3 via interaction at a STAT consensus element in the Cxcl2 promoter. G-CSF coordinately stimulated the association of STAT3, induction of the transcriptionally active H3K4me3 modification, and recruitment of RNA Pol II at the Cxcl2 proximal promoter, as well as the promoter region of Il8rb, encoding the MIP-2 receptor. These results suggest that the G-CSF–STAT3 pathway directly regulates transcriptional events that induce neutrophil mobilization.

Despite the potent ability of dendritic cells (DCs) to stimulate lymphocyte responses and host immunity, granulocyte-macrophage colony-stimulating factor-derived DCs (GM-DCs) used as antitumor vaccines have demonstrated relatively modest success in cancer immunotherapy. We found that injecting GM-DCs into melanoma tumors in mice, or culturing GM-DCs with melanoma-secreted cytokines or melanoma-conditioned medium, rapidly suppressed DC-intrinsic expression of the gene encoding inhibitor of differentiation 2 (ID2), a transcriptional regulator. Melanoma-associated cytokines repressed Id2 transcription in murine DCs through the activation of signal transducer and activator of transcription 3 (STAT3). Enforced expression of ID2 in GM-DCs (ID2-GM-DCs) suppressed their production of the proinflammatory cytokine tumor necrosis factor-α (TNF-α). Vaccination with ID2-GM-DCs slowed the progression of melanoma tumors and enhanced animal survival, which was associated with an increased abundance of tumor-infiltrating interferon-γ-positive CD4(+) effector and CD8(+) cytotoxic T cells and a decreased number of tumor-infiltrating regulatory CD4(+) T cells. The efficacy of the ID2-GM-DC vaccine was improved by combinatorial treatment with a blocking antibody to programmed cell death protein-1 (PD-1), a current immunotherapy that overcomes suppressive immune checkpoint signaling. Collectively, our data reveal a previously unrecognized STAT3-mediated immunosuppressive mechanism in DCs and indicate that DC-intrinsic ID2 promotes tumor immunity by modulating tumor-associated CD4(+) T cell responses. Thus, inhibiting STAT3 or overexpressing ID2 selectively in DCs may improve the efficiency of DC vaccines in cancer therapy.