Atherosclerosis is a leading cause of death worldwide in industrialized countries. Disease progression and regression are associated with different activation states of macrophages derived from inflammatory monocytes entering the plaques. The features of monocyte-to-macrophage transition and the full spectrum of macrophage activation states during either plaque progression or regression, however, are incompletely established. Here, we use a combination of single-cell RNA sequencing and genetic fate mapping to profile, for the first time to our knowledge, plaque cells derived from CX3CR1+ precursors in mice during both progression and regression of atherosclerosis. The analyses revealed a spectrum of macrophage activation states with greater complexity than the traditional M1 and M2 polarization states, with progression associated with differentiation of CXC3R1+ monocytes into more distinct states than during regression. We also identified an unexpected cluster of proliferating monocytes with a stem cell–like signature, suggesting that monocytes may persist in a proliferating self-renewal state in inflamed tissue, rather than differentiating immediately into macrophages after entering the tissue.
Jian-Da Lin, Hitoo Nishi, Jordan Poles, Xiang Niu, Caroline Mccauley, Karishma Rahman, Emily J. Brown, Stephen T. Yeung, Nikollaq Vozhilla, Ada Weinstock, Stephen A. Ramsey, Edward A. Fisher, P’ng Loke
Immunotherapy has emerged as a promising approach to treat cancer. However, partial responses across multiple clinical trials support the significance of characterizing intertumor and intratumor heterogeneity to achieve better clinical results and as potential tools in selecting patients for different types of cancer immunotherapies. Yet, the type of heterogeneity that informs clinical outcome and patient selection has not been fully explored. In particular, the lack of characterization of immune response–related genes in cancer cells hinders the further development of metrics to select and optimize immunotherapy. Therefore, we analyzed single-cell RNA-Seq data from lung adenocarcinoma patients and cell lines to characterize the intratumor heterogeneity of immune response–related genes and demonstrated their potential impact on the efficacy of immunotherapy. We discovered that IFN-γ signaling pathway genes are heterogeneously expressed and coregulated with other genes in single cancer cells, including MHC class II (MHCII) genes. The downregulation of genes in IFN-γ signaling pathways in cell lines corresponds to an acquired resistance phenotype. Moreover, analysis of 2 groups of tumor-restricted antigens, namely neoantigens and cancer testis antigens, revealed heterogeneity in their expression in single cells. These analyses provide a rationale for applying multiantigen combinatorial therapies to prevent tumor escape and establish a basis for future development of prognostic metrics based on intratumor heterogeneity.
Ke-Yue Ma, Alexandra A. Schonnesen, Amy Brock, Carla Van Den Berg, S. Gail Eckhardt, Zhihua Liu, Ning Jiang
The auto antigen (Ag)-specific regulatory T cells (Tregs) from pluripotent stem cells (PSCs), i.e., PSC-Tregs, have the ability to suppress autoimmunity. PSC-Tregs can be programmed to be tissue-associated and to infiltrate into local inflamed tissues to suppress autoimmune responses after adoptive transfer. Nevertheless, the mechanisms by which the auto Ag-specific PSC-Tregs suppress the autoimmune response remain to be fully elucidated. In this study, we generated the functional auto Ag-specific Tregs from the induced PSC (iPSCs), i.e., iPSC-Tregs, and investigated the underlying mechanisms of autoimmunity suppression by these Tregs in a type 1 diabetes (T1D) murine model. A double transgenic (Tg) mouse model of T1D was established in F1 mice in which the first generation of RIP-mOVA Tg mice that were crossed with OT-I T cell receptor (TCR) Tg mice was challenged with vaccinia viruses expressing OVA (VACV-OVA). We show that adoptive transfer of OVA-specific iPSC-Tregs greatly suppressed autoimmunity in the animal model and prevented the insulin-secreting pancreatic β cells from destruction. Further, we demonstrate that the adoptive transfer significantly reduced the expression of ICAM-1 in the diabetic pancreas and inhibited the migration of pathogenic CD8+ T cells and the production of the pro-inflammatory IFN-γ in the pancreas. These results indicate that the stem cell-derived tissue-associated Tregs can robustly accumulate in the diabetic pancreas, and through down-regulating the expression of ICAM-1 in the local inflamed tissues and inhibiting the production of pro-inflammatory cytokine IFN-γ, suppress the migration and activity of the pathogenic immune cells that cause T1D.
Mohammad Haque, Fengyang Lei, Xiaofang Xiong, Jugal Kishore Das, Xingcong Ren, Deyu Fang, Shahram Salek-Ardakani, Jin-Ming Yang, Jianxun Song
Acute respiratory distress syndrome is an often fatal disease that develops after acute lung injury and trauma. How released tissue damage signals, or alarmins, orchestrate early inflammatory events is poorly understood. Herein we reveal that IL-33, an alarmin sequestered in the lung epithelium, is required to limit inflammation after injury due to an unappreciated capacity to mediate Foxp3+ Treg control of local cytokines and myeloid populations. Specifically, Il33–/– mice are more susceptible to lung damage-associated morbidity and mortality that is typified by augmented levels of the proinflammatory cytokines and Ly6Chi monocytes in the bronchoalveolar lavage fluid. Local delivery of IL-33 at the time of injury is protective, but requires the presence of Treg cells. IL-33 stimulates both mouse and human Treg to secrete IL-13. Using Foxp3Cre x Il4/Il13fl/fl mice, we show that Treg expression of IL-13 is required to prevent mortality after acute lung injury by controlling local levels of G-CSF, IL-6, and MCP-1 and inhibiting accumulation of Ly6Chi monocytes. Our study identifies a new regulatory mechanism involving IL-33 and Treg secretion of IL-13 in response to tissue damage that is instrumental in limiting local inflammatory responses and may shape the myeloid compartment after lung injury.
Quan Liu, Gaelen K. Dwyer, Yifei Zhao, Huihua Li, Lisa R. Mathews, Anish Bhaswanth Chakka, Uma R. Chandran, Jake A. Demetris, John F. Alcorn, Keven M. Robinson, Luis A. Ortiz, Bruce Pitt, Angus W. Thomson, Ming-Hui Fan, Timothy R. Billiar, Heth R. Turnquist
B-cells are key contributors to chronic autoimmune pathology in multiple sclerosis (MS). Clonally related B-cells exist in the cerebrospinal fluid (CSF), meninges, and central nervous system (CNS) parenchyma of MS patients. We sought to investigate the presence of clonally related B-cells over time by performing immunoglobulin heavy chain variable region repertoire sequencing on B-cells from longitudinally collected blood and CSF samples of MS patients (n=10). All patients were untreated at the time of the initial sampling; the majority (n=7) were treated with immune modulating therapies 1.2 (+/-0.3 SD) years later during the second sampling. We found clonal persistence of B-cells in the CSF of five patients; these B-cells were frequently immunoglobulin (Ig) class-switched and CD27+. We identified specific blood B-cell subsets that appear to provide input into CNS repertoires over time. We demonstrate complex patterns of clonal B-cell persistence in CSF and blood, even in patients on immune modulating therapy. Our findings support the concept that peripheral B-cell activation and CNS-compartmentalized immune mechanisms can in part therapy-resistant.
Ariele L. Greenfield, Ravi Dandekar, Akshaya Ramesh, Erica L. Eggers, Hao Wu, Sarah Laurent, William Harkin, Natalie S. Pierson, Martin S. Weber, Roland G. Henry, Antje Bischof, Bruce A.C. Cree, Stephen L. Hauser, Michael R. Wilson, H.-Christian von Büdingen
Chimeric antigen receptor (CAR) technology can be used to engineer the antigen-specificity of regulatory T cells (Tregs) and improve their potency as an adoptive cell therapy in multiple disease models. As synthetic receptors, CARs carry the risk of immunogenicity, particularly when derived from non-human antibodies. Using an HLA-A*02:01-specific CAR (A2-CAR) encoding a single-chain Fv derived from a mouse antibody, we developed a panel of 20 humanized (h)A2-CARs. Systematic testing demonstrated variations in expression, ability to bind HLA-A*02:01, and stimulate human Treg suppression in vitro. In addition, we developed a new method to comprehensively map the alloantigen-specificity of CARs, revealing that humanization reduced HLA-A cross reactivity. In vivo bioluminescence imaging showed rapid trafficking and persistence of hA2-CAR Tregs in A2-expressing allografts, with eventual migration to draining lymph nodes. Adoptive transfer of hA2-CAR Tregs suppressed HLA-A2+ cell mediated xenogeneic graft-versus-host disease and diminished rejection of human HLA-A2+ skin allografts. These data provide a platform for systematic development and specificity testing of humanized alloantigen-specific CARs which can be used to engineer specificity and homing of therapeutic Tregs.
Nicholas A.J. Dawson, Caroline Lamarche, Romy E. Hoeppli, Peter Bergqvist, Vivian Fung, Emma McIver, Qing Huang, Jana Gillies, Madeleine Speck, Paul C. Orban, Jonathan W. Bush, Majid Mojibian, Megan K. Levings
Recovery from acute lung injury (ALI) is an active process. Foxp3+ regulatory T cells (Tregs) contribute to recovery from ALI through modulating immune responses and enhancing alveolar epithelial proliferation and tissue repair. The current study investigates Treg transcriptional profiles during resolution of ALI in mice. Tregs from either lung or splenic tissue were isolated from uninjured mice or mice recovering from ALI and then examined for differential gene expression between these conditions. In mice with ALI, Tregs isolated from the lungs had hundreds of differentially expressed transcripts compared to those from the spleen, indicating that organ-specificity and microenvironment are critical in Treg function. These regulated transcripts suggest which intracellular signaling pathways modulate Treg behavior. Interestingly, several transcripts having no prior recognized function in Tregs were differentially expressed by lung Tregs during resolution. Further investigation into two identified transcripts, Mmp12 and Sik1, revealed that Treg-specific expression of each play a role in Treg-promoted ALI resolution. This study provides novel information describing the signals that may expand resident Tregs, recruit or retain them to the lung during ALI, and modulate their function. The results provide insight into both tissue- and immune microenvironment-specific transcriptional differences through which Tregs direct their effects.
Jason R. Mock, Catherine F. Dial, Miriya K. Tune, Dustin L. Norton, Jessica R. Martin, John C. Gomez, Robert S. Hagan, Hong Dang, Claire M. Doerschuk
Epithelial ovarian cancer (EOC) often presents with metastases and ascites. Granulocytic myeloid-derived suppressor cells are an immature population that impairs anti-tumor immunity. Since suppressive granulocytes in the ascites of patients with newly diagnosed EOC were morphologically mature, we hypothesized that PMN were rendered suppressive in the tumor microenvironment. Circulating PMN from patients were not suppressive, but acquired a suppressor phenotype (defined as ≥ 1 log10 reduction of anti-CD3/CD28-stimulated T cell proliferation) after ascites supernatant exposure. Ascites supernatants (20/31) recapitulated the suppressor phenotype in PMN from healthy donors. T cell proliferation was restored with ascites supernatant removal and re-stimulation. PMN suppressors also inhibited T cell activation and cytokine production. PMN suppressors completely suppressed proliferation in naïve, central memory, and effector memory T cells, and in engineered tumor antigen-specific cytotoxic T lymphocytes, while antigen-specific cell lysis was unaffected. Inhibition of complement C3 activation and PMN effector functions, including CR3 signaling, protein synthesis, and vesicular trafficking, abrogated the PMN suppressor phenotype. Moreover, malignant effusions from patients with various metastatic cancers also induced the C3-dependent PMN suppressor phenotype. These results point to PMN impairing T cell expansion and activation in the tumor microenvironment and the potential for complement inhibition to abrogate this barrier to anti-tumor immunity.
Kelly L. Singel, Tiffany R. Emmons, ANM Nazmul H. Khan, Paul C. Mayor, Shichen Shen, Jerry T. Wong, Kayla Morrell, Kevin H. Eng, Jaron Mark, Richard B. Bankert, Junko Matsuzaki, Richard C. Koya, Anna M. Blom, Kenneth R. McLeish, Jun Qu, Sanjay Ram, Kirsten B. Moysich, Scott I. Abrams, Kunle Odunsi, Emese Zsiros, Brahm H. Segal
BACKGROUND. Multiple therapeutic strategies to restore immune regulation and slow type 1 diabetes (T1D) progression are in development and testing. A major challenge has been defining biomarkers to prospectively identify subjects likely to benefit from immunotherapy and/or measure intervention effects. We previously found that compared to healthy controls, Tregs from children with new-onset T1D have an altered Treg gene signature (TGS), suggesting this could be an immunoregulatory biomarker. METHODS. nanoString was used to assess the TGS in sorted Tregs (CD4+CD25hiCD127lo) or Peripheral Blood Mononuclear Cells (PBMC) from individuals with T1D or type 2 diabetes, healthy controls, or T1D recipients of immunotherapy. Biomarker discovery pipelines were developed and applied to various sample group comparisons. RESULTS. Compared to controls, the TGS in isolated Tregs or PBMCs is altered in adult new-onset and cross-sectional T1D cohorts, with sensitivity and specificity of biomarkers increased by including T1D-associated single nucleotide polymorphisms in algorithms. The TGS was distinct in T1D versus type 2 diabetes, indicating disease-specific alterations. TGS measurement at the time of T1D onset revealed an algorithm that accurately predicted future rapid versus slow C-peptide decline, as determined by longitudinal analysis of placebo arms of START and T1DAL trials. The same algorithm stratified participants in a phase I/II clinical trial of ustekinumab (αIL-12/23p40) for future rapid versus slow C-peptide decline. CONCLUSION. These data suggest that biomarkers based on measuring Treg gene signatures could be a new approach to stratify patients and monitor autoimmune activity in T1D.
Anne M. Pesenacker, Virginia Chen, Jana Gillies, Cate Speake, Ashish K. Marwaha, Annika C. Sun, Samuel Chow, Rusung Tan, Thomas Elliott, Jan P. Dutz, Scott J. Tebbutt, Megan K. Levings
Th1 Tregs are characterized by the acquisition of proinflammatory cytokine secretion and reduced suppressor activity. Th1 Tregs are found at increased frequency in autoimmune diseases, including type 1 diabetes and multiple sclerosis (MS). We have previously reported that in vitro stimulation with IL-12 recapitulates the functional and molecular features of MS-associated Th1 Tregs, revealing a central role for hyperactivation of the Akt pathway in their induction. TIGIT is a newly identified coinhibitory receptor that marks Tregs that specifically control Th1 and Th17 responses. Here, we report that signaling through TIGIT counteracts the action of IL-12 in inducing the Th1 program. Specifically, TIGIT signaling represses production of IFN-γ and T-bet expression and restores suppressor function in Tregs treated with IL-12. FoxO1 functional inhibition abolishes the protective effect of TIGIT, indicating that TIGIT signaling promotes FoxO1 nuclear localization. Consistent with this observation, signaling through TIGIT leads to a rapid suppression of Akt function and FoxO1 phosphorylation. Finally, TIGIT stimulation reduces the production of IFN-γ and corrects the suppressor defect of Tregs from patients with MS. Our results indicate an important role for TIGIT in controlling the functional stability of Tregs through repression of Akt, suggesting that the TIGIT pathway could be targeted for immunomodulatory therapies in human autoimmune disorders.
Liliana E. Lucca, Pierre-Paul Axisa, Emily R. Singer, Neal M. Nolan, Margarita Dominguez-Villar, David A. Hafler
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