Even with the prolific clinical use of next-generation cancer therapeutics, many tumors remain unresponsive or become refractory to therapy, creating a medical need. In cancer, DCs are indispensable to T cell activation, so there is a restriction on cytotoxic T cell immunity if DCs are not present in sufficient numbers in the tumor and draining lymph nodes to uptake and present relevant cancer antigens. To address this bottleneck, we developed a Flt3L-based therapeutic named Alb-Flt3L that demonstrated superior pharmacokinetic properties compared to Flt3L, including significantly longer half-life, accumulation in tumor and lymph node, and cross-presenting DCs expansion following a single injection. We demonstrated that Alb-Flt3L, in combination with standard-of-care chemotherapy and radiation therapy, serves as an in situ vaccination strategy capable of engendering polyclonal tumor neoantigen-specific immunity spontaneously. In addition, Alb-Flt3L-mediated tumor control synergized with immune checkpoint blockade delivered as anti-PD-L1. The mechanism of action of Alb-Flt3L treatment revealed a dependency on Batf3, type-I-interferons, and plasmacytoid DCs. Finally, the ability of Alb-Flt3L to expand human DC was explored in humanized mice. We observed significant expansion of human cross-presenting DC subsets, supporting the notion that Alb-Flt3L could be used clinically to modulate human DC populations in future cancer therapeutic regimens.
Brandon Lam, Yu Jui Kung, John Lin, Ssu-Hsueh Tseng, Hsin-Fang Tu, Claire Huang, Brandon Lee, Esteban Velarde, Ya Chea Tsai, Rafael Villasmil, Sung Taek Park, Deyin Xing, Chien-Fu Hung, T.-C. Wu
Matthew D. Taves, Shizuka Otsuka, Jonathan D. Ashwell
Hormone-receptor-positive breast cancer (HR+) is immunologically cold and has not benefited from advances in immunotherapy. In contrast, subsets of triple-negative breast cancer (TNBC) display high leukocytic infiltration and respond to checkpoint blockade. CD8+T cells, the main effectors of anti-cancer responses, recognize MHC I-associated peptides (MAPs). Our work aimed to characterize the repertoire of MAPs presented by HR+ and TNBC tumors. Using mass spectrometry, we identified 57,094 unique MAPs in 26 primary breast cancer samples. MAP source genes highly overlapped between both subtypes (>70%). We identified 25 tumor-specific antigens (TSAs) mainly deriving from aberrantly expressed regions. TSAs were most frequently identified in TNBC samples (70%) and were more shared among TCGA TNBC than HR+ samples. In the TNBC cohort, the predicted number of TSAs positively correlated with leukocytic infiltration (p<0.05) and overall survival (p<0.05), supporting their immunogenicity in vivo. We detected 49 tumor-associated antigens, some of which derived from cancer-associated fibroblasts. Functional expansion of specific T cell assays confirmed the in vitro immunogenicity of several TSAs and TAAs. Our study identified attractive targets for cancer immunotherapy in both breast cancer subtypes. The higher prevalence of TSAs in TNBC tumors provides a rationale for their responsiveness to checkpoint blockade.
Eralda Kina, Jean-Philippe Laverdure, Chantal Durette, Joël Lanoix, Mathieu Courcelles, Qingchuan Zhao, Anca Apavaloaei, Jean-David Larouche, Marie-Pierre Hardy, Krystel Vincent, Patrick Gendron, Leslie Hesnard, Catherine Thériault, Maria Virginia Ruiz Cuevas, Grégory Ehx, Pierre Thibault, Claude Perreault
Regulatory T cells (Tregs) are instrumental in maintaining immune tolerance and preventing destructive autoimmunity, but how heterogeneous Treg populations are established remains largely unknown. Here, we show that Zfp335 deletion in Tregs failed to differentiate into effector Tregs (eTregs) and lose Treg-suppressive function and that KO mice exhibited early-onset lethal autoimmune inflammation with unrestricted activation of conventional T cells. Single-cell RNA-Seq analyses revealed that Zfp335-deficient Tregs lacked a eTreg population and showed dramatic accumulation of a dysfunctional Treg subset. Mechanistically, Zfp335-deficient Tregs displayed reduced oxidative phosphorylation and dysfunctional mitochondrial activity. Further studies revealed that Zfp335 controlled eTreg differentiation by regulating fatty acid oxidation (FAO) through direct targeting of the FAO enzyme Hadha. Importantly, we demonstrate a positive correlation between ZNF335 and HADHA expression in human eTregs. Our findings reveal that Zfp335 controls FAO-driven eTreg differentiation to establish immune tolerance.
Xin Wang, Lina Sun, Biao Yang, Wenhua Li, Cangang Zhang, Xiaofeng Yang, Yae Sun, Xiaonan Shen, Yang Gao, Bomiao Ju, Yafeng Gao, Dan Liu, Jiapeng Song, Xiaoxuan Jia, Yanhong Su, Anjun Jiao, Haiyan Liu, Lianjun Zhang, Lan He, Lei Lei, WanJun Chen, Baojun Zhang
Tissue-resident lymphocytes provide organ-adapted protection against invading pathogens. Whereas their biology has been examined in great detail in various infection models, their generation and functionality in response to vaccination has not been comprehensively analyzed in humans. We therefore studied SARS-CoV2 mRNA-vaccine-specific T cells in surgery specimens of kidney, liver, lung, bone marrow and spleen in comparison to paired blood samples from largely virus-naïve individuals. As opposed to lymphoid tissues, non-lymphoid organs harbored significantly elevated frequencies of Spike-specific CD4+ T cells compared to blood showing hallmarks of tissue residency and an expanded memory pool. Organ-derived CD4+ T cells further exhibited increased polyfunctionality over those detected in blood. Single-cell RNA sequencing together with T cell receptor repertoire analysis indicated that the clonotype rather than organ origin is a major determinant of transcriptomic state in vaccine-specific CD4+ T cells. In summary, our data demonstrate that SARS-CoV2 vaccination entails acquisition of tissue memory and residency features in organs distant from the inoculation site, thereby contributing to our understanding of how local tissue protection might be accomplished.
Vanessa Proß, Arne Sattler, Söeren Lukassen, Laura Tóth, Linda Marie Laura Thole, Janine Siegle, Carolin Stahl, An He, Georg Damm, Daniel Seehofer, Christina Götz, Christian Bayerl, Pia Jäger, Alexander Macke, Stephan Eggeling, Bernadette Kirzinger, Thomas Mayr, Hermann Herbst, Katharina Beyer, Dominik Laue, Jan Krönke, Jan Braune, Friederike Rosseck, Beatrice Kittner, Frank Friedersdorff, Mandy Hubatsch, Sarah Weinberger, Nils Lachmann, Veit Maria Hofmann, Eva Schrezenmeier, Carolin Ludwig, Hubert Schrezenmeier, Katharina Jechow, Christian Conrad, Katja Kotsch
The functional integrity of Treg cells is interwoven with cellular metabolism; however, the mechanisms governing Treg cell metabolic programs remain elusive. Here, we identified that the deubiquitinase USP47 inhibited RNA m6A reader YTHDF1-mediated c-Myc translation to maintain Treg cell metabolic and functional homeostasis. USP47 positively correlated with the tumor-infiltrating Treg cell signature in colorectal cancer and gastric cancer patient samples. USP47 ablation compromised Treg cell homeostasis and function in vivo, resulting in the development of inflammatory disorders, and boosted antitumor immune responses. USP47 deficiency in Treg cells triggered the accumulation of the c-Myc protein and in turn exacerbated hyperglycolysis. Mechanistically, USP47 prevented YTHDF1 ubiquitination to attenuate the association of YTHDF1 with translation initiation machinery, thereby decreasing m6A-based c-Myc translation efficiency. Our findings reveal that USP47 directs m6A-dependent metabolic programs to orchestrate Treg cell homeostasis and suggest novel approaches for selective immune modulation in cancer and autoimmune diseases by targeting USP47.
Aiting Wang, Haiyan Huang, Jian-Hong Shi, Xiaoyan Yu, Rui Ding, Yuerong Zhang, Qiaoqiao Han, Zhi-Yu Ni, Xia Li, Ren Zhao, Qiang Zou
BACKGROUND. Macrophage activation syndrome (MAS) is a life-threatening complication of Still’s disease (SD) characterized by overt immune cell activation and cytokine storm. We aimed to further understand the immunologic landscape of SD and MAS. METHOD. We profiled peripheral blood mononuclear cells (PBMC) from healthy controls and patients with SD with or without MAS using bulk RNA sequencing (RNA-seq) and single-cell RNA-seq (scRNA-seq). We validated and expanded the findings by mass cytometry, flow cytometry and in vitro studies. RESULTS. Bulk RNA-seq of PBMC from patients with SD-associated MAS revealed strong expression of genes associated with type I interferon (IFN-I) signaling and cell proliferation, in addition to the expected IFN-γ signal, compared to healthy controls and SD patients without MAS. scRNA-seq analysis of > 65,000 total PBMC confirmed IFN-I and IFN-γ signatures and localized the cell proliferation signature to cycling CD38+HLA-DR+ cells within CD4+ T cell, CD8+ T cell and NK cell populations. CD38+HLA-DR+ lymphocytes exhibited prominent IFN-g production, glycolysis, and mTOR signaling. Cell-cell interaction modeling suggested a network linking CD38+HLA-DR+ lymphocytes with monocytes through IFN-γ signaling. Notably, the expansion of CD38+HLA-DR+ lymphocytes in MAS was greater than in other systemic inflammatory conditions in children. In vitro stimulation of PBMC demonstrated that IFN-I and IL-15 – both elevated in MAS patients – synergistically augmented the generation of CD38+HLA-DR+ lymphocytes, while Janus kinase inhibition mitigated this response. CONCLUSION. MAS associated with SD is characterized by overproduction of IFN-I, which may act in synergy with IL-15 to generate CD38+HLA-DR+ cycling lymphocytes that produce IFN-γ.
Zhengping Huang, Kailey E. Brodeur, Liang Chen, Yan Du, Holly Wobma, Evan E. Hsu, Meng Liu, Joyce C. Chang, Margaret H. Chang, Janet Chou, Megan Day-Lewis, Fatma Dedeoglu, Olha Halyabar, James A. Lederer, Tianwang Li, Mindy S. Lo, Meiping Lu, Esra Meidan, Jane W. Newburger, Adrienne G. Randolph, Mary Beth F. Son, Robert P. Sundel, Maria L. Taylor, Huaxiang Wu, Qing Zhou, Scott W. Canna, Kevin Wei, Lauren A. Henderson, Peter A. Nigrovic, Pui Y. Lee
Monocytes and monocyte-derived macrophages (MDM) from blood circulation infiltrate glioblastoma (GBM) and promote growth. Here we show that PDGFB-driven GBM cells induce the expression of the potent pro-inflammatory cytokine IL-1β in MDM, which engages IL-1R1 in tumor cells, activates the NF-kB pathway, and subsequently leads to induction of monocyte chemoattractant proteins (MCPs). Thus, a feedforward paracrine circuit of IL-1β/IL-1R1 between tumors and MDM creates an interdependence driving PDGFB-driven GBM progression. Genetic loss or locally antagonizing IL-1β/IL-1R1 leads to reduced MDM infiltration, diminished tumor growth, reduced exhausted CD8+ T cells, and thereby extends the survival of tumor-bearing mice. In contrast to IL-1β, IL-1α exhibits anti-tumor effects. Genetic deletion of Il1a/b is associated with decreased recruitment of lymphoid cells and loss of interferon signaling in various immune populations and subsets of malignant cells and is associated with decreased survival time of PDGFB-driven tumor-bearing mice. In contrast to PDGFB-driven GBM, Nf1-silenced tumors have a constitutively-active NF-kB pathway, which drives the expression of MCPs to recruit monocytes into tumors. These results indicate local antagonism of IL-1β could be considered as an effective therapy specifically for proneural GBM.
Zhihong Chen, Bruno Giotti, Milota Kaluzova, Montserrat Puigdelloses Vallcorba, Kavita Rawat, Gabrielle Price, Cameron J. Herting, Gonzalo Piñero, Simona Cristea, James L. Ross, James Ackley, Victor Maximov, Frank Szulzewsky, Wes Thomason, Mar Marquez-Ropero, Angelo Angione, Noah Nichols, Nadejda M. Tsankova, Franziska Michor, Dmitry M. Shayakhmetov, David H. Gutmann, Alexander M. Tsankov, Dolores Hambardzumyan
The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in macrophages, but whether they have distinct functions remains unclear. We confirmed that GLUT1 expression increased after M1 polarization stimuli and found that GLUT3 expression increased after M2 stimulation in macrophages. Conditional deletion of Glut3 (LysM-Cre Glut3fl/fl) impaired M2 polarization of bone marrow derived macrophages. Alternatively activated macrophages from the skin of atopic dermatitis patients showed increased GLUT3 expression, and a calcipotriol-induced model of atopic dermatitis was rescued LysM-Cre Glut3fl/fl mice. M2-like macrophages expressed GLUT3 in human wound tissues as assessed by transcriptomics and co-staining, and GLUT3 expression was significantly decreased in non-healing, compared with healing, diabetic foot ulcers. In an excisional wound healing model, LysM-Cre Glut3fl/fl mice showed significantly impaired M2 macrophage polarization and delayed wound healing. GLUT3 promoted IL-4/STAT6 signaling, independent from its glucose transport activity. Unlike plasma membrane-localized GLUT1, GLUT3 was localized primarily to endosomes and was required for the efficient endocytosis of IL4Ra subunits. GLUT3 interacted directly with GTP-bound RAS in vitro and in vivo through its intracytoplasmic loop domain (ICH), and this interaction was required for efficient STAT6 activation and M2 polarization. PAK activation and macropinocytosis were also impaired without GLUT3, suggesting broader roles for GLUT3 in the regulation of endocytosis. Thus, GLUT3 is required for efficient alternative macrophage polarization and function, through a glucose transport-independent, RAS-mediated role in the regulation of endocytosis and IL-4/STAT6 activation.
Dong-Min Yu, Jiawei Zhao, Eunice E. Lee, Dohun Kim, Ruchika Mahapatra, Elysha K. Rose, Zhiwei Zhou, Calvin R. Hosler, Abdullah El-Kurdi, Jun-yong Choe, E. Dale Abel, Gerta Hoxhaj, Kenneth D. Westover, Raymond J. Cho, Jeffrey B. Cheng, Richard C. Wang
Prashant Rai, Martin Sharpe, Charan K. Ganta, Paul J. Baker, Katrin D. Mayer-Barber, Brian E. Fee, Gregory A. Taylor, Michael B. Fessler