Therapeutic IL-12 has demonstrated the ability to reduce local immune suppression in preclinical models, but clinical development has been limited by severe inflammation-related adverse events with systemic administration. Here, we show that potent immunologic tumor control of established syngeneic carcinomas can be achieved by i.t. administration of a tumor-targeted IL-12 antibody fusion protein (NHS–rmIL-12) using sufficiently low doses to avoid systemic toxicity. Single-cell transcriptomic analysis and ex vivo functional assays of NHS–rmIL-12–treated tumors revealed reinvigoration and enhanced proliferation of exhausted CD8+ T lymphocytes, induction of Th1 immunity, and a decrease in Treg number and suppressive capacity. Similarly, myeloid cells transitioned toward inflammatory phenotypes and displayed reduced suppressive capacity. Cell type–specific IL-12 receptor–KO BM chimera studies revealed that therapeutic modulation of both lymphoid and myeloid cells is required for maximum treatment effect and tumor cure. Study of single-cell data sets from human head and neck carcinomas revealed IL-12 receptor expression patterns similar to those observed in murine tumors. These results describing the diverse mechanisms underlying tumor-directed IL-12–induced antitumor immunity provide the preclinical rationale for the clinical study of i.t. NHS–IL-12.
Youji Hong, Yvette Robbins, Xinping Yang, Wojciech K. Mydlarz, Anastasia Sowers, James B. Mitchell, James L. Gulley, Jeffrey Schlom, Sofia R. Gameiro, Cem Sievers, Clint T. Allen
Understanding the endogenous mechanisms regulating resolution of pain may identify novel targets for treatment of chronic pain. Resolution of chemotherapy-induced peripheral neuropathy (CIPN) after treatment completion depends on CD8+ T cells and on IL-10 produced by other cells. Using Rag2–/– mice lacking T and B cells and adoptive transfer of Il13–/– CD8+ T cells, we showed that CD8+ T cells producing IL-13 were required for resolution of CIPN. Intrathecal administration of anti–IL-13 delayed resolution of CIPN and reduced IL-10 production by dorsal root ganglion macrophages. Depleting local CD206+ macrophages also delayed resolution of CIPN. In vitro, TIM3+CD8+ T cells cultured with cisplatin, apoptotic cells, or phosphatidylserine liposomes produced IL-13, which induced IL-10 in macrophages. In vivo, resolution of CIPN was delayed by intrathecal administration of anti-TIM3. Resolution was also delayed in Rag2–/– mice reconstituted with Havcr2 (TIM3)–/– CD8+ T cells. Our data indicated that cell damage induced by cisplatin activated TIM3 on CD8+ T cells, leading to increased IL-13 production, which in turn induced macrophage IL-10 production and resolution of CIPN. Development of exogenous activators of the IL-13/IL-10 pain resolution pathway may provide a way to treat the underlying cause of chronic pain.
Susmita K. Singh, Karen Krukowski, Geoffroy O. Laumet, Drew Weis, Jenolyn F. Alexander, Cobi J. Heijnen, Annemieke Kavelaars
Phosphopeptides derived from dysregulated protein phosphorylation in cancer cells can be processed and presented by MHC class I and class II molecules and, therefore, represent an untapped class of tumor-specific antigens that could be used as widely expressed “public” cancer neoantigens (NeoAgs). We generated a TCR mimic (TCRm) mAb, 6B1, specific for a phosphopeptide derived from insulin receptor substrate 2 (pIRS2) presented by HLA-A*02:01. The pIRS2 epitope’s presentation by HLA-A*02:01 was confirmed by mass spectrometry. The TCRm 6B1 specifically bound to pIRS2/HLA-A2 complex on tumor cell lines that expressed pIRS2 in the context of HLA-A*02:01. Bispecific mAbs engaging CD3 of T cells were able to kill tumor cell lines in a pIRS2- and HLA-A*02:01–restricted manner. Structure modeling shows a prerequisite for an arginine or lysine at the first position to bind mAb. Therefore, 6B1 could recognize phosphopeptides derived from various phosphorylated proteins with similar amino acid compositions. This raised the possibility that a TCRm specific for the pIRS2/HLA-A2 complex could target a range of phosphopeptides presented by HLA-A*02:01 in various tumor cells. This is the first TCRm mAb to our knowledge targeting a phosphopeptide/MHC class I complex; the potential of this class of agents for clinical applications warrants further investigation.
Tao Dao, Sung Soo Mun, Zaki Molvi, Tatyana Korontsvit, Martin G. Klatt, Abdul G. Khan, Elisabeth K. Nyakatura, Mary Ann Pohl, Thomas E. White, Paul J. Balderes, Ivo C. Lorenz, Richard J. O’Reilly, David A. Scheinberg
The intermittent fasting (IF) diet has profound benefits for diabetes prevention. However, the precise mechanisms underlying IF’s beneficial effects remain poorly defined. Here, we show that the expression levels of cyclooxygenase-2 (COX-2), an enzyme that produces prostaglandins, are suppressed in white adipose tissue (WAT) of obese humans. In addition, the expression of COX-2 in WAT is markedly upregulated by IF in obese mice. Adipocyte-specific depletion of COX-2 led to reduced fractions of CD4+Foxp3+ Tregs and a substantial decrease in the frequency of CD206+ macrophages, an increase in the abundance of γδT cells in WAT under normal chow diet conditions, and attenuation of IF-induced antiinflammatory and insulin-sensitizing effects, despite a similar antiobesity effect in obese mice. Mechanistically, adipocyte-derived prostaglandin E2 (PGE2) promoted Treg proliferation through the CaMKII pathway in vitro and rescued Treg populations in adipose tissue in COX-2–deficient mice. Ultimately, inactivation of Tregs by neutralizing anti-CD25 diminished IF-elicited antiinflammatory and insulin-sensitizing effects, and PGE2 restored the beneficial effects of IF in COX-2–KO mice. Collectively, our study reveals that adipocyte COX-2 is a key regulator of Treg proliferation and that adipocyte-derived PGE2 is essential for IF-elicited type 2 immune response and metabolic benefits.
Chunqing Wang, Xing Zhang, Liping Luo, Yan Luo, Xin Yang, Xiaofeng Ding, Lu Wang, Huyen Le, Lily Elizabeth R. Feldman, Xuebo Men, Cen Yan, Wendong Huang, Yingmei Feng, Feng Liu, Xuexian O. Yang, Meilian Liu
Recent data establish a logarithmic expansion of leucine rich repeat containing G protein coupled receptor 5–positive (Lgr5+) colonic epithelial stem cells (CESCs) in human colorectal cancer (CRC). Complementary studies using the murine 2-stage azoxymethane–dextran sulfate sodium (AOM-DSS) colitis-associated tumor model indicate early acquisition of Wnt pathway mutations drives CESC expansion during adenoma progression. Here, subdivision of the AOM-DSS model into in vivo and in vitro stages revealed DSS induced physical separation of CESCs from stem cell niche cells and basal lamina, a source of Wnt signals, within hours, disabling the stem cell program. While AOM delivery in vivo under non-adenoma-forming conditions yielded phenotypically normal mucosa and organoids derived thereof, niche injury ex vivo by progressive DSS dose escalation facilitated outgrowth of Wnt-independent dysplastic organoids. These organoids contained 10-fold increased Lgr5+ CESCs with gain-of-function Wnt mutations orthologous to human CRC driver mutations. We posit CRC originates by niche injury–induced outgrowth of normally suppressed mutated stem cells, consistent with models of adaptive oncogenesis.
Stefan Klingler, Kuo-Shun Hsu, Guoqiang Hua, Maria Laura Martin, Mohammad Adileh, Timour Baslan, Zhigang Zhang, Philip B. Paty, Zvi Fuks, Anthony M.C. Brown, Richard Kolesnick
Parturition is a well-orchestrated process characterized by increased uterine contractility, cervical ripening, and activation of the chorioamniotic membranes; yet, the transition from a quiescent to a contractile myometrium heralds the onset of labor. However, the cellular underpinnings of human parturition in the uterine tissues are still poorly understood. Herein, we performed a comprehensive study of the human myometrium during spontaneous term labor using single-cell RNA sequencing (scRNA-Seq). First, we established a single-cell atlas of the human myometrium and unraveled the cell type–specific transcriptomic activity modulated during labor. Major cell types included distinct subsets of smooth muscle cells, monocytes/macrophages, stromal cells, and endothelial cells, all of which communicated and participated in immune (e.g., inflammation) and nonimmune (e.g., contraction) processes associated with labor. Furthermore, integrating scRNA-Seq and microarray data with deconvolution of bulk gene expression highlighted the contribution of smooth muscle cells to labor-associated contractility and inflammatory processes. Last, myometrium-derived single-cell signatures can be quantified in the maternal whole-blood transcriptome throughout pregnancy and are enriched in women in labor, providing a potential means of noninvasively monitoring pregnancy and its complications. Together, our findings provide insights into the contributions of specific myometrial cell types to the biological processes that take place during term parturition.
Roger Pique-Regi, Roberto Romero, Valeria Garcia-Flores, Azam Peyvandipour, Adi L. Tarca, Errile Pusod, Jose Galaz, Derek Miller, Gaurav Bhatti, Robert Para, Tomi Kanninen, Ola Hadaya, Carmen Paredes, Kenichiro Motomura, Jeffrey R. Johnson, Eunjung Jung, Chaur-Dong Hsu, Stanley M. Berry, Nardhy Gomez-Lopez
Chronic obstructive pulmonary disease (COPD) is a debilitating chronic disease and the third-leading cause of mortality worldwide. It is characterized by airway neutrophilia, promoting tissue injury through release of toxic mediators and proteases. Recently, it has been shown that neutrophil-derived extracellular vesicles (EVs) from lungs of patients with COPD can cause a neutrophil elastase–dependent (NE-dependent) COPD-like disease upon transfer to mouse airways. However, in vivo preclinical models elucidating the impact of EVs on disease are lacking, delaying opportunities for therapeutic testing. Here, we developed an in vivo preclinical mouse model of lung EV–induced COPD. EVs from in vivo LPS-activated mouse neutrophils induced COPD-like disease in naive recipients through an α-1 antitrypsin–resistant, NE-dependent mechanism. Together, these results show a key pathogenic and mechanistic role for neutrophil-derived EVs in a mouse model of COPD. Broadly, the in vivo model described herein could be leveraged to develop targeted therapies for severe lung disease.
Camilla Margaroli, Matthew C. Madison, Liliana Viera, Derek W. Russell, Amit Gaggar, Kristopher R. Genschmer, J. Edwin Blalock
The G protein–coupled CXC chemokine receptor 4 (CXCR4) is a candidate therapeutic target for tissue fibrosis. A fully human single-domain antibody-like scaffold i-body AD-114-PA600 (AD-114) with specific high binding affinity to CXCR4 has been developed. To define its renoprotective role, AD-114 was administrated in a mouse model of renal fibrosis induced by folic acid (FA). Increased extracellular matrix (ECM) accumulation, macrophage infiltration, inflammatory response, TGF-β1 expression, and fibroblast activation were observed in kidneys of mice with FA-induced nephropathy. These markers were normalized or partially reversed by AD-114 treatment. In vitro studies demonstrated AD-114 blocked TGF-β1–induced upregulated expression of ECM, matrix metalloproteinase-2, and downstream p38 mitogen-activated protein kinase (p38 MAPK) and PI3K/AKT/mTOR signaling pathways in a renal proximal tubular cell line. Additionally, these renoprotective effects were validated in a second model of unilateral ureteral obstruction using a second generation of AD-114 (Fc-fused AD-114, also named AD-214). Collectively, these results suggest a renoprotective role of AD-114 as it inhibited the chemotactic function of CXCR4 as well as blocked CXCR4 downstream p38 MAPK and PI3K/AKT/mTOR signaling, which establish a therapeutic strategy for AD-114 targeting CXCR4 to limit renal fibrosis.
Qinghua Cao, Chunling Huang, Hao Yi, Anthony J. Gill, Angela Chou, Michael Foley, Chris G. Hosking, Kevin K. Lim, Cristina F. Triffon, Ying Shi, Xin-Ming Chen, Carol A. Pollock
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease characterized by collagen deposition within the lung interstitium. Bacterial infection is associated with increased morbidity and more rapid mortality in IPF patient populations, and pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) are commonly isolated from the lungs of hospitalized patients with IPF. Despite this, the effects of fibrotic lung injury on critical immune responses to infection remain unknown. In the present study, we show that, like humans with IPF, fibrotic mice infected with MRSA exhibit increased morbidity and mortality compared with uninfected fibrotic mice. We determine that fibrosis conferred a defect in MRSA clearance compared with nonfibrotic mice, resulting from blunted innate immune responses. We show that fibrosis inhibited neutrophil intracellular killing of MRSA through impaired neutrophil elastase release and oxidative radical production. Additionally, we demonstrate that lung macrophages from fibrotic mice have impaired phagocytosis of MRSA. Our study describes potentially novel impairments of antimicrobial responses upon pulmonary fibrosis development, and our findings suggest a possible mechanism for why patients with IPF are at greater risk of morbidity and mortality related to infection.
Helen I. Warheit-Niemi, Summer J. Edwards, Shuvasree SenGupta, Carole A. Parent, Xiaofeng Zhou, David N. O’Dwyer, Bethany B. Moore
Why multisystem inflammatory syndrome in children (MIS-C) develops after SARS-CoV-2 infection in a subset of children is unknown. We hypothesized that aberrant virus–specific T cell responses contribute to MIS-C pathogenesis. We quantified SARS-CoV-2–reactive T cells, serologic responses against major viral proteins, and cytokine responses from plasma and peripheral blood mononuclear cells in children with convalescent COVID-19, in children with acute MIS-C, and in healthy controls. Children with MIS-C had significantly lower virus-specific CD4+ and CD8+ T cell responses to major SARS-CoV-2 antigens compared with children convalescing from COVID-19. Furthermore, T cell responses in participants with MIS-C were similar to or lower than those in healthy controls. Serologic responses against spike receptor binding domain (RBD), full-length spike, and nucleocapsid were similar among convalescent COVID-19 and MIS-C, suggesting functional B cell responses. Cytokine profiling demonstrated predominant Th1 polarization of CD4+ T cells from children with convalescent COVID-19 and MIS-C, although cytokine production was reduced in MIS-C. Our findings support a role for constrained induction of anti–SARS-CoV-2–specific T cells in the pathogenesis of MIS-C.
Vidisha Singh, Veronica Obregon-Perko, Stacey A. Lapp, Anna Marie Horner, Alyssa Brooks, Lisa Macoy, Laila Hussaini, Austin Lu, Theda Gibson, Guido Silvestri, Alba Grifoni, Daniela Weiskopf, Alessandro Sette, Evan J. Anderson, Christina A. Rostad, Ann Chahroudi
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